Cellular targeted pharmaceutically active substance or label delivery system

ABSTRACT

The present invention relates to an isolated cellular targeted delivery system comprising a CD45+ leukocyte cell comprising within said cell a complex of one or more iron binding proteins and an active pharmaceutically active substance and/or label as well as methods for producing such isolated cellular targeted delivery system and uses of such system for prophylaxis, therapy, diagnosis or theragnosis, in particular for prophylactic or therapeutic vaccination, therapy of cancer, particularly metastatic cancer or inflammatory diseases.

The present invention relates to an isolated cellular targeted deliverysystem comprising a CD45⁺ leukocyte cell comprising within said cell acomplex of one or more iron binding proteins and an activepharmaceutically active substance and/or label as well as methods forproducing such isolated cellular targeted delivery system and uses ofsuch system for prophylaxis, therapy, diagnosis or theragnosis, inparticular for prophylactic or therapeutic vaccination, therapy ofcancer, particularly metastatic cancer or inflammatory diseases.

BACKGROUND OF THE INVENTION

Current imaging tools are capable of detecting large metastases (largerthan 0.5-1 cm in size). However, they rarely detect the early spread ofmetastatic tumour cells. Human metastases smaller than 0.5 cm areavascular so without proper blood and oxygen supply. It means that thedelivery of contrast agents through the blood circulation for thepurpose of labelling these metastases and imaging them is not possible.The presence of hypoxia is a common characteristic of micrometastaseswhere hypoxic fraction may be as high as 90% with little or no bloodperfusion (Li, et al. 2012, Journal of Solid Tumours, 2(2): 28-33).Thus, severe hypoxia is considered as a general feature ofmicro-metastases.

The targeting of one or more micrometastases hidden within a largepopulation of normal cells presents a unique challenge since access tothe micrometastases is impeded by several bio-barriers, poor bloodsupply, further obstacles are presented by small size of themicrometastases and their dispersion to organs.

For the same reason micrometastases are often refractive to therapy.While the solid tumours from which the micrometastases have originatedoften respond well to conventional therapy there is often regrowth atthe site of the primary tumour or at sites of metastasis. Thisconstitutes a serious problem in clinical oncology (Muthana, et al.2012, Cancer Res; 73(2); 490-495). It is related to characteristics ofthe microenvironment of solid tumours that limit drug penetration,thereby exposing the tumour to lower than efficacious concentrations ofdrugs (Hobbs, et al. 1998, Proc Natl Acad Sci USA: 4607-4612). This iscaused by inadequate vasculature resulting in: high heterogeneity ofcancer cells, low oxygen tension (hypoxia), low pH and low glucoseconcentration within the mass (Kizaka-Kondoh, et al., 2003, Cancer Sci94(12):1021-1028). Additionally, rapid tumour cell proliferation in someareas might outpace the rate of new blood vessel growth, promotingformation of hypoxic area (Lewis and Murdoch, 2005, Am J Pathol167(3):627-635). This abnormal vessel architecture and, subsequently,their impaired function resulting in tumour hypoxia is associated with amore malignant phenotype and poor survival in patients suffering fromsolid tumours and results in both treatment failure due to decreaseddrug uptake and hypoxiainducible changes in cancer cells (Sun, et al.,2012, Clin Cancer Res 18(3):758-770; Sullivan, et al., 2008 Mol CancerTher 7(7):1961-1973; Kizaka-Kondoh, et al., 2003, Cancer Sci94(12):1021-1028). Moreover, chemotherapy or radiotherapy causesadditional formation of large areas of tumour hypoxia thus making thetreatment of tumour even more difficult. The fact that the efficacy ofanticancer therapy is limited by the presence of hypoxic tumour cellshas resulted in the introduction of variety of therapeutic approachesaimed at overcoming this problem.

The present inventors have discovered that CD45⁺ leukocyte cells, inparticular activated macrophages, their precursors monocytes,lymphocytes and granulocytes can uptake pharmaceutically activesubstances, labels or pharmaceutically active substances and labelscomplexed with one or more iron binding proteins in vitro and deliverthese complexes to or into cells, preferably to or into tumour cells invivo or other cells undergoing stress (e.g. oxidative stress). Based onthis observation the present inventors have overcome one or more of theabove stated problems of the prior art. Thus, the targeted deliverysystem of the present invention provides inta alia one or more of thefollowing advantages: (i) specific delivery of one or morepharmaceutically active substances, labels or pharmaceutically activesubstances and labels to tissues that attract above mentioned CD45⁺leukocytes, preferably into diseased cells, (ii) protection ofpharmaceutically active substances, labels or pharmaceutically activesubstances and labels from inactivation in the blood circulation orclearance from the body, (iii) delivery of pharmaceutically activesubstances, labels or pharmaceutically active substances and labels to,preferably into cells of poorly or non-vascularized areas of disease,e.g. metastases, hypoxic areas within larger tumours, rheumatic lesions,inflamed lymph nodes, avascular wounds, skin, (iv) reduced toxicity ofpharmaceutically active substances, labels or pharmaceutically activesubstances and labels, (v) delivery of pharmaceutically activesubstances, labels or pharmaceutically active substances and labels withpoor pharmacokinetics, (vi) reduced side effects of the drugs due totheir targeted delivery, (vii) higher treatment efficacy with lowerdoses of the drugs due to targeted delivery; and/or (viii) lower risk oflocal tissue injury at the site of drug administration due toadministration of the drug linked with iron-binding protein, which isloaded inside the CD45⁺ leukocyte; and/or (ix) possibility to detecthighly hypoxic small metastases; and/or (x) early detection ofinflammatory diseases.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to an isolated targeteddelivery system comprising a CD45⁺ monocyte-macrophage, CD45⁺lymphocyte, in particular CD45⁺ natural killer cells (NK cells),precursors of any of these cell types, preferably MDSC, and/or CD45⁺granulocyte, (“CD45⁺ leukocyte cell”), or their precursors, preferablyMDSC, comprising within said cell a complex of one or more iron bindingproteins and a pharmaceutically active substance, label orpharmaceutically active substance and label.

In a second aspect the present invention relates to an isolated targeteddelivery label system comprising a CD45⁺ leukocyte cell, preferablycapable of being labelled, comprising one or more labels, preferablyradiolabels or their conjugates and combinations.

In a third aspect the present invention relates to a method ofpreparation of the isolated targeted delivery system of claims 1 to 25comprising steps of

-   -   a) providing purified iron binding protein;    -   b) covalently or non-covalently linking a pharmaceutically        active substance, label or pharmaceutically active substance and        label to and/or encapsulating a pharmaceutically active        substance, label or pharmaceutically active substance and label        in an iron binding protein;    -   c) providing a CD45⁺ leukocyte cell; and    -   d1) incubating the CD45⁺ leukocyte cell in the presence of the        iron binding protein produced in step b) until the CD45⁺        leukocyte cell is at least partially loaded with the complex of        the iron binding protein and the a pharmaceutically active        substance, label or pharmaceutically active substance and label        produced in step b); and/or    -   d2) incubating CD45⁺ leukocyte cell in the presence of the label        until the CD45⁺ leukocyte cell is at least partially labelled        with the label.

In a fourth aspect the present invention relates to an isolated targeteddelivery system of the first aspect or second aspect of the invention orproducible according to the method of the third aspect of the inventionfor use as a medicament or diagnostic.

In a fifth aspect the present invention relates to a pharmaceutical ordiagnostic composition comprising the isolated targeted delivery systemof the first or second aspect of the present invention or producibleaccording to the method of the third aspect of the invention and apharmaceutically acceptable carrier and/or suitable excipient(s).

In a sixth aspect the present invention relates to an isolated targeteddelivery system of the first aspect or second aspect of the presentinvention or producible according to the method of the third aspect ofthe invention for use in preventing, treating or diagnosing a diseasecharacterized by hypoxic areas within the diseases tissue and/or byareas of oxidative stress; tumours, preferably solid tumours and/or itsmetastases, preferably breast cancer, pancreatic cancer, bladder cancer,lung cancer, colon cancer, or a tumour having hypoxic areas, aninflammatory disease, inflamed tissue, preferably inflamed joints orarthritic joints, inflamed lung, inflamed intestine or other inflamedtissue; lymph nodes, preferably inflamed lymph nodes, or othernon-physiological lymph nodes that develop during disease preferably butnot only during infection, cancer, or autoimmune disease; or ischemicareas, in particular in skin wounds or after organ infarctus (heart) orischemic retina or for prophylactic or therapeutic vaccination, inparticular to prevent or treat an infectious disease or cancer. Thisaspect also includes also of antigens to physiological ornon-physiological lymph nodes in order to vaccinate an individual or toinduce immune memory.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Definitions

To practice the present invention, unless otherwise indicated,conventional methods of chemistry, biochemistry, and recombinant DNAtechniques are employed which are explained in the literature in thefield (cf., e.g., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press,Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps. Asused in this specification and the appended claims, the singular forms“a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

The term “targeted pharmaceutically active substance delivery” refers tothe delivery of a pharmaceutically active substance to a patient whichresults in an increased concentration of the pharmaceutically activesubstance in a particular region of the body when compared to otherregions of the body of that patient. Preferably, the relativeconcentrations are compared between the diseased region(s) of the bodyand other regions of the body having similar access to the bloodcirculation. In preferred embodiments the concentration of thepharmaceutically active substance in a given number of cells or a givenbiopsy volume from the diseased region is at least 10% higher, ifcompared to the identical number of cells or biopsy volume from anon-diseased region after administration of the targetedpharmaceutically active substance delivery system of the presentinvention, preferably after 2-24 hrs. More preferably, the concentrationof the pharmaceutically active substance in the diseased region of thebody of a patient is at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70% at least 80%, at least 90%, at least100%, at least 150%, at least 200%, at least 250%, at least 300%, atleast 350%, at least 400%, at least 450%, at least 500%, more preferablyat least 1000% higher than in a non-diseased region of the body afteradministration of the targeted pharmaceutically active substancedelivery system of the present invention, preferably after 2-24 hrs.When assessed on the basis of total body distribution it is preferredthat at least 5% of the pharmaceutically active substance administeredto a patient is delivered to the diseased region of the body, preferablyat least 10%, more preferably at least 15%. The targeted delivery of thepharmaceutically active substance limits the potential deleteriouseffects of a pharmaceutically active substance to the diseased region ofthe body.

The term “targeted label delivery” or “targeted contrast agent delivery”refers to the delivery of a label, in particular a contrast agent to apatient or a person to be diagnosed which results in an increasedconcentration of the label, in particular the contrast agent in aparticular region of the body when compared to other regions of the bodyof that patient. Preferably, the relative concentrations are comparedbetween the diseased region(s) of the body and other regions of the bodyhaving similar access to the blood circulation. In preferred embodimentsthe concentration of the label, in particular a contrast agent in agiven number of cells or a given biopsy volume from the diseased regionis at least 10% higher, if compared to the identical number of cells orbiopsy volume from a non-diseased region after administration of thetargeted delivery system of the present invention, preferably after 1-24hrs. More preferably, the concentration of the label, in particular thecontrast agent in the diseased region of the body of a patient is atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70% at least 80%, at least 90%, at least 100%, at least 150%, atleast 200%, at least 250%, at least 300%, at least 350%, at least 400%,at least 450%, at least 500%, more preferably at least 1000% higher thanin a non-diseased region of the body after administration of thetargeted delivery system of the present invention, preferably after 2-24hrs. When assessed on the basis of total body distribution it ispreferred that at least 5% of the label, in particular the contrastagent administered to a patient or a person to be diagnosed is deliveredto the diseased region of the body, preferably at least 10%, morepreferably at least 15%. The targeted delivery of the label, inparticular the contrast agent limits the potential deleterious effectsof an label, in particular the contrast agent to the diseased region ofthe body.

The term “targeted theragnostic delivery” has a similar meaning as setout above for “targeted pharmaceutically active substance” and “targetedlabel delivery, however instead of only delivering a pharmaceuticalactive substance or a label the complex of the iron binding proteincomprises in this embodiment both a pharmaceutically active substanceand a label, thereby allowing the concomitant delivery of both apharmaceutically active substance and a label.

The term “targeted pharmaceutically active substance delivery system” isused in the present application to refer to a system that is capable ofdelivering a pharmaceutically active substance to the targeted region,i.e. capable of targeted delivery within the body of a patient,preferably to a diseased region.

The term “targeted label delivery system” is used in the presentapplication to refer to a system that is capable of delivering a labelto the targeted region, i.e. capable of targeted delivery within thebody of a patient, preferably to a diseased region.

The term “targeted theragnostic delivery system” is used in the presentapplication to refer to a system that is capable of delivering a complexof a pharmaceutically active substance and at the same time a label tothe targeted region, i.e. capable of targeted delivery within the bodyof a patient, preferably to a diseased region and thus allowssimultaneous treatment and diagnosis and/or treatment monitoring.

The term “targeted delivery system” is used to commonly refer to“targeted pharmaceutically active substance delivery system”, “targetedlabel delivery system” and “targeted theragnostic delivery system”

The term “pharmaceutically active substance” is used in the context ofthe present invention to refer to a substance that modifies or modulatescell activity, preferably a cell activity that leads to a disease in apatient. The term encompasses both substances which are alreadypharmaceutically active or which are capable of being activated, i.e. aprodrug. Examples of such pharmaceutically active substances include socalled (i) “small molecules”, (ii) polynucleotides and (iii) peptide orproteins. The term “small molecule” is used in the context of thepresent invention to refer to a hydrocarbon with a molecular mass ofbelow 1.500 g/mol or to pharmaceutically active radioactive isotopes.Preferred, drugs that can be used comprise anti-cancer drugs,pharmaceutically active radioactive isotopes or ferrihydrite.

The term “prodrug” as used in the context of the present inventionrefers to any active ingredient that, after administration, ismetabolized or otherwise converted to a biologically active or moreactive ingredient (or drug) with respect to at least one property. Incomparison to the drug, a prodrug is modified chemically in a mannerthat makes it, relative to the drug, less active or inactive, but thechemical modification is such that the corresponding drug is generatedby metabolic or other biological processes after the prodrug isadministered to the patient. A prodrug may for example have, relative tothe active drug, altered metabolic stability or transportcharacteristics, fewer side effects or lower toxicity, or improvedflavor (for example, see the reference Nogrady, 1985, MedicinalChemistry A Biochemical Approach, Oxford University Press, New York,pages 388-392, incorporated herein by reference). A prodrug may besynthesized using reactants other than the corresponding drug.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and are understood as a polymeric or oligomeric macromoleculemade from nucleotide monomers. Nucleotide monomers are composed of anucleobase, a five-carbon sugar (such as but not limited to ribose or2′-deoxyribose), and one to three phosphate groups. Typically, apolynucleotide is formed through phosphodiester bonds between theindividual nucleotide monomers. In the context of the present inventionreferred to nucleic acid molecules include but are not limited toribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixturesthereof such as e.g. RNA-DNA hybrids. The nucleic acids, can e.g. besynthesized chemically, e.g. in accordance with the phosphotriestermethod (see, for example, Uhlmann, E. & Peyman, A. (1990) ChemicalReviews, 90, 543-584). “Aptamers” are nucleic acids which bind with highaffinity to a polypeptide. Aptamers can be isolated by selection methodssuch as SELEmir146-a (see e.g. Jayasena (1999) Clin. Chem., 45, 1628-50;Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107; U.S. Pat. No.5,582,981) from a large pool of different single-stranded RNA molecules.Aptamers can also be synthesized and selected in their mirror-imageform, for example as the L-ribonucleotide (Nolte et al. (1996) Nat.Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat. Biotechnol., 14,1112-5). Forms which have been isolated in this way enjoy the advantagethat they are not degraded by naturally occurring ribonucleases and,therefore, possess greater stability.

The term “peptide” or “polypeptide” is used interchangeably in thecontext of the present invention to refer to a chain of at least twoamino acids linked by peptide bonds. Thus, the term “peptide” in thecontext of the present invention is also used to refer to amino acidchains with more than 50, more than 100 or more than 150 amino acids.

The term “antibody” as used in the context of the present inventionrefers to a glycoprotein belonging to the immunoglobulin superfamily;the terms antibody and immunoglobulin are often used interchangeably. Anantibody refers to a protein molecule produced by plasma cells and isused by the immune system to identify and neutralize foreign objectssuch as bacteria and viruses. The antibody recognizes a unique part ofthe foreign target, its antigen.

The term “antibody fragment” as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Examples of binding fragments encompassed within the term“antibody fragment” include a fragment antigen binding (Fab) fragment, aFab′ fragment, a F(ab′)₂ fragment, a heavy chain antibody, asingle-domain antibody (sdAb), a single-chain fragment variable (scFv),a fragment variable (Fv), a V_(H) domain, a V_(L) domain, a singledomain antibody, a nanobody, an IgNAR (immunoglobulin new antigenreceptor), a di-scFv, a bispecific T-cell engager (BITEs), a dualaffinity re-targeting (DART) molecule, a triple body, a diabody, asingle-chain diabody, an alternative scaffold protein, and a fusionprotein thereof.

The term “diabody” as used within this specification refers to a fusionprotein or a bivalent antibody which can bind different antigens. Adiabody is composed of two single protein chains which comprisefragments of an antibody, namely variable fragments. Diabodies comprisea heavy chain variable domain (V_(H)) connected to a light-chainvariable domain (V_(L)) on the same polypeptide chain (V_(H)-V_(L), orV_(L)-V_(H)). By using a short peptide connecting the two variabledomains, the domains are forced to pair with the complementary domain ofanother chain and thus, create two antigen-binding sites. Diabodies cantarget the same (monospecific) or different antigens (bispecific).

A “single domain antibody”, refers to antibody fragments consisting of asingle, monomeric variable domain of an antibody. Simply, they onlycomprise the monomeric heavy chain variable regions of heavy chainantibodies produced by camelids or cartilaginous fish. Due to theirdifferent origins they are also referred to VHH or VNAR (variable newantigen receptor)-fragments. Alternatively, single-domain antibodies canbe obtained by monomerization of variable domains of conventional mouseor human antibodies by the use of genetic engineering. They show amolecular mass of approximately 12-15 kDa and thus, are the smallestantibody fragments capable of antigen recognition. Further examplesinclude nanobodies or nanoantibodies.

The term “antibody mimetic” as used within the context of the presentspecification refers to compounds which can specifically bind antigens,similar to an antibody, but are not structurally related to antibodies.Usually, antibody mimetics are artificial peptides or proteins with amolar mass of about 3 to 20 kDa which comprise one, two or more exposeddomains specifically binding to an antigen. Examples include inter aliathe LACI-D1 (lipoprotein-associated coagulation inhibitor); affilins,e.g. human-γ B crystalline or human ubiquitin; cystatin; Sac7D fromSulfolobus acidocaldarius; lipocalin and anticalins derived fromlipocalins; DARPins (designed ankyrin repeat domains); SH3 domain ofFyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10^(th)type III domain of fibronectin; adnectins: knottins (cysteine knotminiproteins); atrimers; evibodies, e.g. CTLA4-based binders,affibodies, e.g. three-helix bundle from Z-domain of protein A fromStaphylococcus aureus; Trans-bodies, e.g. human transferrin;tetranectins, e.g. monomeric or trimeric human C-type lectin domain;microbodies, e.g. trypsin-inhibitor-II; affilins; armadillo repeatproteins. Nucleic acids and small molecules are sometimes consideredantibody mimetics as well (aptamers), but not artificial antibodies,antibody fragments and fusion proteins composed from these. Commonadvantages over antibodies are better solubility, tissue penetration,stability towards heat and enzymes, and comparatively low productioncosts.

The term “antigen” is used to refer to a substance, preferably animmunogenic peptide that comprises at least one epitope, preferably anepitope that elicits a B or T cell response or B cell and T cellresponse.

An “epitope”, also known as antigenic determinant, is that part of asubstance, e.g. an immunogenic polypeptide, which is recognized by theimmune system. Preferably, this recognition is mediated by the bindingof antibodies, B cells, or T cells to the epitope in question. In thiscontext, the term “binding” preferably relates to a specific binding.Epitopes usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. The term “epitope” comprises bothconformational and non-conformational epitopes. Conformational andnon-conformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

An immunogenic polypeptide according to the present invention is,preferably, derived from a pathogen selected from the group consistingof viruses, bacteria and protozoa. In particular embodiments. However,in an alternative embodiment of the present invention the immunogenicpolypeptide is a tumor antigen, i.e. polypeptide or fragment of apolypeptide specifically expressed by a cancer.

The term “sequence identity” is used throughout the specification withregard to polypeptide and nucleotide sequence comparisons. In case wheretwo sequences are compared and the reference sequence is not specifiedin comparison to which the sequence identity percentage is to becalculated, the sequence identity is to be calculated with reference tothe longer of the two sequences to be compared, if not specificallyindicated otherwise. If the reference sequence is indicated, thesequence identity is determined on the basis of the full length of thereference sequence indicated by SEQ ID, if not specifically indicatedotherwise. For example, a polypeptide sequence consisting of 200 aminoacids compared to a reference 300 amino acid long polypeptide sequencemay exhibit a maximum percentage of sequence identity of 66.6% (200/300)while a sequence with a length of 150 amino acids may exhibit a maximumpercentage of sequence identity of 50% (150/300). If 15 out of those 150amino acids are different from the respective amino acids of the 300amino acid long reference sequence, the level of sequence identitydecreases to 45%. The similarity of nucleotide and amino acid sequences,i.e. the percentage of sequence identity, can be determined via sequencealignments. Such alignments can be carried out with several art-knownalgorithms, preferably with the mathematical algorithm of Karlin andAltschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) orwith the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T.J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. onhttp://www.ebi.ac.uk/Tools/clustalw/ or onhttp://www.ebi.ac.uk/Tools/clustalw2/index.html or onhttp://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html.Preferred parameters used are the default parameters as they are set onhttp://www.ebi.ac.uk/Tools/clustalw/ orhttp://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequenceidentity (sequence matching) may be calculated using e.g. BLAST, BLAT orBlastZ (or BlastX). BLAST protein searches are performed with the BLASTPprogram, score=50, word length=3. To obtain gapped alignments forcomparative purposes, Gapped BLAST is utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programsare used. Sequence matching analysis may be supplemented by establishedhomology mapping techniques like Shuffle-LAGAN (Brudno M.,Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields.Structure based alignments for multiple protein sequences and/orstructures using information from sequence database searches, availablehomologs with 3D structures and user-defined constraints may also beused (Pei J, Grishin N V: PROMALS: towards accurate multiple sequencealignments of distantly related proteins. Bioinformatics 2007,23:802-808; 3DCoffee@igs: a web server for combining sequences andstructures into a multiple sequence alignment. Poirot O, Suhre K,Abergel C, O'Toole E, Notredame C. Nucleic Acids Res. 2004 Jul. 1;32:W37-40). When percentages of sequence identity are referred to in thepresent application, these percentages are calculated in relation to thefull length of the longer sequence, if not specifically indicatedotherwise.

The term “label” as used in the context of the present invention refersto any kind of compound being suitable for diagnostic purposes.Preferred compounds are selected from a fluorescent dye, a radioisotopeand a contrast agent. A contrast agent is a dye or other substance thathelps to show abnormal areas inside the body. In one embodiment the termlabel refers to a compound that comprises a chelating agent which formsa complex with divalent or trivalent metal cations. Preferredradioisotopes/fluorescence emitting isotopes are selected from the groupconsisting of alpha radiation emitting isotopes, gamma radiationemitting isotopes, Auger electron emitting isotopes, X-ray emittingisotopes, fluorescent isotopes, such as ⁶⁵Tb, fluorescence emittingisotopes, such as ¹⁸F, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ^(99m)Tc, ¹⁴⁰La, ¹⁷⁵Yb,¹⁵³Sm, ¹⁶⁶Ho, ⁸⁸Y, ⁸⁹Zr, ⁹⁰Y, ¹⁴⁹Pm, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi,⁷²As, ⁷²Se, ⁹⁷Ru, ¹⁰⁹Pd, ¹⁰⁵Rh, ^(101m15)Rh, ¹¹⁹Sb, ¹²⁸Ba, ¹²³I, ¹²⁴I,¹³¹I, ¹⁹⁷Hg, ²¹¹At, ¹⁶⁹Eu, ²⁰³Pb, ²¹²Pb, ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁹⁸Auand ¹⁹⁹Ag as well as conjugates, compounds comprising such isotopes andcombinations of above with proteins, peptides, small molecularinhibitors, antibodies or other compounds, e.g. ¹⁸F fluorodeoxyglucose(¹⁸F-FDG), ⁸⁹Zr-oxide or ⁶⁴Cu-porfirin. Preferred fluorescent dyes areselected from the following classes of dyes: Xanthens (e.g.Fluorescein), Acridines (e.g. Acridine Yellow), Oxazines (e.g. Oxazine1), Cynines (e.g. Cy7/Cy 3), Styryl dyes (e.g. Dye-28), Coumarines (e.g.Alexa Fluor 350), Porphines (e.g. Chlorophyll B), Metal-Ligand-Complexes(e.g. PtOEPK), Fluorescent proteins (e.g APC, R-Phycoerythrin),Nanocrystals (e.g QuantumDot 705), Perylenes (e.g. Lumogen Red F300) andPhtalocyanines (e.g. IRDYE™700DX) as well as conjugates and combinationsof these classes of dyes or fluorescent ⁶⁵Tb emitting. Preferredcontrast agents are selected from paramagnetic agents, e.g. Gd, Eu, Wand Mn, preferably complexed with a chelating agent. Further options aresuperparamagnetic iron (Fe) complexes and particles, compoundscontaining atoms of high atomic number, i.e. iodine for computertomography (CT), microbubbles and carriers such as liposomes thatcontain these contrast agents.

The term “leukocyte” is used in the context of the present invention torefer to cells of the immune system that are involved in protecting thebody against both infectious disease and foreign invaders. Allleukocytes are produced and derived from multipotent cells in the bonemarrow known as a hematopoietic stem cells. Leukocytes are foundthroughout the body, including the blood and lymphatic system. Allleukocytes have nuclei, which distinguishes them from the other bloodcells, the anucleated red blood cells (RBCs) and platelets. Types ofleukocyte can be classified in standard ways. Two pairs of the broadestcategories classify them either by structure (granulocytes oragranulocytes) or by cell division lineage (myeloid cells or lymphoidcells). These broadest categories can be further divided into the fivemain types: neutrophils, eosinophils, basophils, lymphocytes, andmonocytes. These types are distinguished by their physical andfunctional characteristics. Monocytes and neutrophils are phagocytic.Further subtypes can be classified; for example, among lymphocytes,there are B cells, T cells, and NK cells. Granulocytes are distinguishedfrom agranulocytes by their nucleus shape (lobed versus round, that is,polymorphonuclear versus mononuclear) and by their cytoplasm granules(present or absent, or more precisely, visible on light microscopy ornot thus visible). The other dichotomy is by lineage: Myeloid cells(neutrophils, monocytes, eosinophils and basophils) are distinguishedfrom lymphoid cells (lymphocytes) by hematopoietic lineage (cellulardifferentiation lineage).

The present inventors have observed that CD45⁺ expression ischaracteristic of a subgroup of leukocyte cells, i.e. monocyte,monocyte-macrophages, lymphocytes, granulocytes, NK cells that aresuitable to be used in the context of the targeted delivery system ofthe present invention, in particular since CD45⁺ leukocyte cells areattracted to particular tissues and cells within the body and arecapable of delivering complexes of one or more iron binding proteins andone or more pharmaceutically active substances, labels orpharmaceutically active substances and labels to or into cells. Thissubgroup of leukocytes is in the following referred to as “CD45⁺leukocyte cells” or “CD45⁺ leukocytes”. Preferably the monocyte is not adendritic cell which differentiation is controlled by one or more of thefollowing transcription factors: IFN-regulatory factor 8 (IRF8), nuclearfactor interleukin (IL)-3-regulated protein (NFIL3), basic leucinezipper transcriptional factor ATF-like 3 (BATF3) or Transcription FactorRelB (NF-KB Subunit)-RELB, Spi-1 Proto-Oncogene (PU/1), recombiningbinding protein suppressor of hairless (RBPJ), IFN-regulatory factor 4(IRF4) or transcription factor E2-2 (also known as (TCF4).

It is understood by the skilled person that CD45⁺ leukocyte cells asdefined above unless of clonal origin are a mixed population ofdifferent leukocytes which share the common property of expressing CD45⁺surface antigen. Accordingly, subpopulations of cells within the diversegroup of CD45⁺ leukocyte cells as defined above are characterizedthroughout the specification by further functional and/or structuralcharacteristics. The term “CD45⁺” indicates that the majority of cellswithin a population of cells or essentially all cells express the CD45⁺surface antigen. In this context and also with reference to othercellular surface antigens, the term “expresses” indicates that thesurface antigen is produced within the cell and detectably exposed onthe surface of a cell. The level of expression and, thus the number ofsurface antigens detectably exposed on the surface of a cell can varygreatly among different leukocytes. Generally, a cell is considered tobe positive, i.e. is indicated to be “+”, for a cellular surfaceantigen, if at least 5, preferably at least 10 copies of the surfaceantigen are detectably exposed on the surface of the cell. The skilledperson is well aware of how to detect, quantify and select for cells,which are positive (or negative) for a given cellular surface antigen.Preferred methods include Fluorescence Activated Cell Sorting (FACS). Inthis technology fluorescently labelled antibodies are used to bind tocellular surface antigens of a population of cells, the cells aresubsequently isolated into single cells and based on fluorescenceintensity measured for the single cell, characterized as being positiveor negative for the given cellular surface antigen. In some embodimentsof the present invention it is indicated that the expression of a givenprotein is high or low. This means that the protein is detectablyexpressed in both instances, i.e. is “+”, however, at different levels.High and low expression, respectively, will mean different absolutenumbers of proteins per cell for different proteins. Thus, a givenprotein may be considered to be expressed at high levels if there aremore than 500 detectable copies of that protein per cell and to beexpressed at low levels if there are between 1 to 50 detectable copiesof that protein per cell. However, another protein may be considered tobe expressed at high levels, if there are more than 5000 detectablecopies and expressed at low levels, if there are between 1 to 500detectable copies per cell. It is well known in the art how to quantifythe number of proteins expressed or produced in a cell using flowcytometry and Becton Dickinson Quantibrite™ bead method (see e.g. Pannu,K. K., 2001, Cytometry. 2001 Dec. 1; 45(4):250-8) or mass spectrometry(see, e.g. Milo, R., 2013, Bioessays, 35(12): 1050-1055). For thepurpose of the present invention the term “high expression” of a givenprotein refers to detectable expression of that protein that is at least70% of the highest expression level found, i.e. number of copies percell, in a population of healthy CD45⁺ leukocytes. The term “lowexpression” of a given protein refers to detectable expression of thatprotein that is 30% or less of the highest expression level found, i.e.number of copies of that protein per cell, in a population of healthyCD45⁺ leukocytes. Preferably, the “highest expression level” isdetermined as the average of the highest expression levels found inhealthy CD45⁺ leukocytes of different subjects. In some embodimentspreferred subpopulations of cells are characterized as “producing” agiven protein. This is understood to mean that the protein is notnecessarily detectable on the surface of the cell but may only bepresent inside the cell. The skilled person is well aware how to detectand/or quantify production of a protein inside a cell and/or selectcells producing such proteins. Alternatively, cell populations can bedefined by expression of specific transcription factors. It is wellknown in the art how to determine expression of a given protein or itsencoding mRNA in a population of cells or even in single cells, e.g.using in vivo labeling with antibodies, FISH assays, in vivo singlemolecule fluorescent microscopy (Crawford, R. et al. Biophys J. (2013)105(11): 2439) alone or in combination with Fluorescent Activated CellSorting (FACS), or by the PrimeFlow technique (e Bioscience), (Adam S.Venable, et. al., (2015) Methods in Molecular Biology).

The term “differentiated monocyte” is used in the context of the presentinvention to refer to a monocyte differentiated from the committedprecursor termed macrophage-DC precursor (MDP) mainly resident in bonemarrow (but could be also in the spleen) and differentiate into eitherdendritic cells or macrophages. In mice they consist of two mainsubpopulations: (i) CD11b⁺ cell with high expression of CX3CR1, lowexpression of CCR2 and Ly6C⁻ and (ii) CD11b⁺ cell with low expression ofCX3CR1, high expression of CCR2 and Ly6C⁺. After leaving the bonemarrow, mouse Ly6C⁺ monocytes differentiate into Ly6C⁻ monocytes incirculation. Similarly, in human monocyte differentiation, it isaccepted that CD14⁺⁺ classical monocytes leave bone marrow anddifferentiate into CD14⁺⁺CD16⁺ intermediate monocytes and sequentiallyto CD14⁺CD16⁺⁺ non-classical monocytes in peripheral blood circulation(Yang et al. 2014; Biomark Res 2(1) doi. 10.1186/2050-7771-2-1).Preferably the differentiated monocyte is not a dendritic cell, whichdifferentiation is controlled by one or more of the followingtranscription factors: IRF8, NFIL3, BATF3, RELB, PU/1, RBPJ, IIRF4,and/or TCF4, and more preferably is not a dendritic cell.

Macrophages are tissue-resident professional phagocytes andantigen-presenting cells (APC), which differentiate from circulatingperipheral blood monocytes (PBMs). The term “activated macrophage” isused in the context of the present invention to refer to any macrophagethat is polarized. Macrophage activation is in general achieved byincubation with interleukins, cytokines and/or growth factors. Inparticular IL-4 and M-CSF can be used as activating agents. Activatedmacrophages of different phenotypes are classified into M1-macrophages,classically activated macrophages (CAM) and M2-macrophages,alternatively activated macrophages (AAM). The classically activatedM1-macrophages comprise immune effector cells with an acute inflammatoryphenotype. These are highly aggressive against bacteria and producelarge amounts of lymphokines (Murray, and Wynn, 2011, J Leukoc Biol,89(4):557-63). The alternatively activated, anti-inflammatoryM2-macrophages can be separated into at least three subgroups. Thesesubtypes have various different functions, including regulation ofimmunity, maintenance of tolerance and tissue repair/wound healing. Theterm “M1 inducer” is used in the context of the present invention torefer to a compound that directs differentiation of PBMs to macrophagesof the M1 type. The term “M2 inducer” is used in the context of thepresent invention to refer to a compound that directs differentiation ofPBMs to macrophages of the M2 type. The skilled person is aware of alarge number of ways to promote differentiation into either M1 or M2macrophages.

The term “phagocytosis by macrophages” is the process by which amacrophage engulfs a solid particle to form an internal vesicle known asa phagosome.

The term “iron binding protein” as used refers to a protein thatnon-covalently binds an iron ion. Examples of such proteins compriseferritin, haemoglobin, transferrin; and lactoferrin. Iron bindingproteins are bound by cellular surface receptors which facilitate theinternalization of these proteins into cells.

In a first aspect the present invention relates to an isolated targeteddelivery system comprising a CD45⁺ monocyte, CD45⁺ monocyte-macrophage,CD45⁺ lymphocyte, CD45⁺ NK cell and/or CD45⁺ granulocyte (hereinafterreferred to as “CD45⁺ leukocyte cell”) comprising within said cell acomplex of one or more iron binding proteins and a pharmaceuticallyactive substance, label or pharmaceutically active substance and label.Preferably the CD45⁺ monocyte is not a dendritic cell, whichdifferentiation is controlled by one or more of the followingtranscription factors: IRF8, NFIL3, BATF3, RELB, PU/1, RBPJ, IIRF4,and/or TCF4, and more preferably is not a dendritic cell.

It has been surprisingly found by the present inventors that CD45⁺leukocyte cells, preferably activated macrophages, preferably M1macrophages, more preferably M2 macrophages acquire pharmaceuticallyactive substances, labels or pharmaceutically active substances andlabels and deliver the pharmaceutically active substances, labels orpharmaceutically active substances and labels in the amount sufficient(i) to be detected using imaging systems to allowing them to be used totrace their location in the body. In case of ¹⁸F-FDG this wasparticularly preferable in case of monocytes and activated monocytes,macrophages, activated macrophages, preferably M1 macrophages and mostpreferably M2 macrophages. Administration of cells-loaded with a label(¹⁸F-FDG, ⁸⁹Zr-oxide), preferably radioactive label can visualize organwith accumulation of the labelled cells preferably by positron emissiontomography (PET) imaging, (ii) to elicit a prophylactic or therapeuticeffect or to allow a therapy and diagnosis of a disease at the sametime.

In a second aspect the present invention relates to an isolated targetedlabel delivery system comprising a CD45⁺ leukocyte cell, preferablycapable of being labelled with one or more labels, preferablyradiolabels or their conjugates and combinations. It is a preferredembodiment of the second aspect of the present invention that the cellis directly linked to or labelled with label. The label may be withinsaid cell, or on its surface, preferably it is on the surface of thecell.

The following preferred embodiments in each case further specify boththe first and second aspect of the present invention.

The ability of a given CD45⁺ leukocyte cell or cell population tointernalize iron binding proteins depends on the expression of receptorsinvolved in this internalization process. Receptors that lead tointernalization of ferritin comprise, e.g. TfR, CXCR4, CD163, and TIM-2.The skilled person is well aware how to measure the amount of uptake ofan iron binding protein and preferred method of measuring the uptake aredescribed in the Example Section below. The present inventors also notedthat subpopulations of CD45⁺ leukocyte cells have a certain propensityto internalize one iron binding protein over another iron bindingprotein and, thus can attain higher complex concentrations and/or showless leakage of the complex from the cells. Such CD45⁺ leukocytesubpopulations are described in more detail below.

The phrase “complex of one or more iron binding proteins and apharmaceutically active substance, label or pharmaceutically activesubstance and label” as used in the context of the present inventionrefers to a composition in which one or more molecules of thepharmaceutically active substance, label or pharmaceutically activesubstance and label are covalently or non-covalently bound to one ormore iron binding proteins. The covalent or non-covalent binding betweenthe one or more iron binding proteins and the one more pharmaceuticallyactive substance, label or pharmaceutically active substance and labelcan be direct or indirect. In the latter case the pharmaceuticallyactive substance, label or pharmaceutically active substance and labelis linked to the iron binding protein via a linker or spacer. Linker orspacers are known to the skilled artisan, such as polyalanine,polyglycin, carbohydrates, (CH₂)n groups or polypeptide linkers. Theskilled artisan will, thus, be able to select the respective suitablelinker(s) or spacer(s) depending on the respective application. If theiron binding proteins form cages like, e.g. ferritin, than the term“complex” also encompasses the enclosure of pharmaceutically activesubstances, labels or pharmaceutically active substances and labelswithin the cage even in the absence of a covalent or non-covalent bondbetween the protein(s) and the active compound(s). The formation of thecomplex allows the transport of the pharmaceutically active substance,label or pharmaceutically active substance and label into the cell whenthe cell is internalizing the iron binding protein. Thus, it ispreferred that the pharmaceutically active substances, labels orpharmaceutically active substances and labels are bound to the ironbinding protein in a way that does not interfere with the transportmechanism. This can be easily tested by the skilled person using uptakeassays known in the art and described in the Example Section below. Itis preferred that the complex is sufficiently stable to survive thetransport within the cell to the target region within the body. Thus, itis preferred that the complex rather than the pharmaceutically activesubstance, label or pharmaceutically active substance and label alone isdelivered to the cells or into the cells in the target region. Thisproperty also reduces possible deleterious effects, e.g. cytotoxicity,of the pharmaceutically active substance, label or pharmaceuticallyactive substance and label to the CD45⁺ leukocyte cell. Ifpharmaceutically active substances, labels or pharmaceutically activesubstances and labels are covalently coupled to the iron bindingproteins such coupling is preferably through amino acids residues knownto be located in surface areas that are not involved in binding to thecellular receptors required for cellular uptake of the iron bindingproteins. Iron binding proteins used in the context of the presentinvention can form stable non-covalently bound complexes with a widevariety of pharmaceutically active substances, labels orpharmaceutically active substances and labels. If the pharmaceuticallyactive substance or label is a peptide, e.g. an antigenic peptide it ispreferred that it is not expressed as a fusion with the iron bindingprotein, since in this case release of the peptide from the iron bindingprotein will require endosomal processing of the entire iron bindingprotein peptide fusion protein.

The CD45⁺ leukocyte cells originate from the patient to be treated insuch case the cell loaded with the complex would be autologous to thepatient. It is also envisioned that patients are MHC typed prior totreatment with the targeted delivery of the present invention and thatthe leukocyte cell type used for a given patient is MHC matched to thepatient. In these two preferred embodiments the CD45⁺ leukocyte cell isa primary cell or derived by a low number of differentiation steps froma primary cell. Alternatively, the CD45⁺ leukocyte cell may be from animmortalized but preferably non-transformed CD45⁺ leukocyte cell line.Thus the blood used for CD45⁺ leukocyte cells, i.e. CD45⁺ monocyte,CD45⁺ monocyte-macrophage, CD45⁺ granulocyte, or CD45⁺ lymphocyte, inparticular CD45⁺ NK cell, isolation is preferably obtained from thepatient to be treated or from a healthy donor. Alternatively the bloodcan be obtained from the blood bank. Use of umbilical cord blood is alsoconsidered herein.

The present inventors noted that a subpopulation of CD45⁺ leukocytecells, which are producible from a CD34⁺ hematopoietic precursor cellare particular suitable for target specific delivery of thepharmaceutically active substance, label or pharmaceutically activesubstance and label. Accordingly, it is preferred that the leukocytesused to produce the target delivery system are derived from CD34⁺hematopoietic precursor cells. The skilled person is well aware how toselect CD34⁺ hematopoietic precursor cells and how to differentiate suchcells into leukocytes.

As indicated above, the term “CD45⁺ leukocyte cell” is used throughoutthis specification to refer to a CD45⁺ monocyte, CD45⁺monocyte-macrophage, CD45⁺ lymphocyte and/or CD45⁺. Preferably, themonocyte is not a dendritic cell, which differentiation is controlled byone or more of the following transcription factors: IRF8, NFIL3, BATF3,RELB, PU/1, RBPJ, IIRF4, and/or TCF4, and more preferably is not adendritic cell. Preferred subpopulations in these general categories ofleukocytes are defined in the following by structural parameters, e.g.presence or absence of a given protein, functional properties and/ormethod of their production/differentiation. As outlined above, thetargeted delivery system of the present invention still provides theadvantages outlined above, if in a population of cells not every cellhas a particular property in as long as the majority of cells withinthat population has that property. Thus, in the following the propertyof one preferred cell of the targeted delivery system of the presentinvention is described. It is appreciated by the skilled person that apharmaceutical composition of the present invention will comprisesmillions of cells and that not every cell within the population willhave the functional and/or structural properties outlined herein butthat the pharmaceutical composition can nevertheless be used to treat adisease, if the majority of cells share the respective functional and/orstructural properties.

The present inventors have recognized that subpopulations of CD45⁺leukocyte cells have particular advantageous properties including amongothers efficiency and/amount of complex uptake in general, ability toretain the complex within the cell, i.e. to avoid leakage and of targetrelease of the pharmaceutically active substance, label orpharmaceutically active substance and label, efficiency of uptake of aparticular iron binding protein and/or targeting to particular tissuesor cells and, thus suitability to treat or ameliorate a particulardisease. The present inventors have, e.g. observed that CD45⁺ leukocytecells, which express one or more of the following antigens: CD204,CD206, CD200R, CCR2 have a preference for ferritin uptake over theuptake of other iron binding proteins. Thus, if the iron binding proteinin the complex is ferritin it is preferred to select CD45⁺ leukocytecells that express one or more of the following antigens: CD204, CD206,CD200R, CCR2. Accordingly, in a preferred embodiment of the presentinvention

-   (i) the monocyte is a CD11b⁺ monocyte, preferably selected from the    group consisting of a CD11b⁺ CD14⁺ monocyte, a CD11b⁺ CD16⁺    monocyte, a CD11b⁺ CD14⁺ CD16⁺ monocyte, a CD11b⁺ CD14⁺ MHCII⁺    monocyte, a CD11b⁺ CD14⁺ CD115⁺ monocyte, CD11b⁺ CD114⁺ monocyte,    CD11b⁺ CD116⁺ monocyte, CD11b⁺ CCR1⁺ monocyte, CD11b⁺ CCR2⁺    monocyte, CD11b⁺ CX3CR⁺ monocyte, CD11b⁺ CXR4⁺ monocyte, CD11b⁺    CXR6⁺ monocyte and a CD11b⁺ CD14⁺ CD33⁺ monocyte, preferably the    monocyte is not a dendritic cell, which differentiation is    controlled by one or more of the following transcription factors:    IRF8, NFIL3, BATF3, RELB, PU/1, RBPJ, IIRF4, and/or TCF4, and more    preferably is not a dendritic cell;-   (ii) the differentiated monocyte or monocyte-macrophage is    differentiated by M-CSF and selected from the group consisting of a    macrophage, an activated macrophage, preferably a CD11b⁺ macrophage,    more preferably a CD11b⁺ CD16⁺ macrophage, CD11b⁺ CD32⁺ macrophage,    CD11b⁺ CD64⁺ macrophage, CD11b⁺ CD68⁺ macrophage, preferably a    CD11b⁺ CD86⁺ M1 macrophage, preferably producing inducible nitric    oxide synthetase (iNOS) and/or secreting interleukin 12 (IL-12) or    preferably CD11b⁺ CCR2⁺ M2 macrophage, CD11b⁺ CD204⁺ M2 macrophage,    CD11b⁺ CD206⁺ M2 macrophage, CD11b⁺ CD204⁺ CD206⁺ M2 macrophage,    CD11b⁺ Major Histocompatibility Complex II⁺ (MHCII⁺) (low or hi    expression) M2 macrophage, CD11b⁺ CD200R⁺ M2 macrophage, CD11b⁺    CD163⁺ M2 macrophage or activated macrophage producing and/or    secreting Arginase-1 and/or interleukin 10 (IL10); preferably the    differentiated monocyte is not a foam cell expressing Lectin-like    oxidized low-density lipoprotein receptor-1 (Lox1⁺), C—X—C chemokine    receptor type 7 (CXCR7⁺) and Nuclear factor (erythroid-derived    2)-like 2 (NRF2⁺). A foam cell is a type of macrophage that localize    to fatty deposits on blood vessel walls, where they ingest    low-density lipoproteins and become loaded with lipids giving them a    foamy appearance. These cells secrete various substances involved in    plaque growth and their death promotes inflammation, thereby    contributing to cardiovascular disease;-   (iii) monocyte-macrophage or activated monocyte-macrophage is    differentiated by M-CSF and is preferably expressing at least one    chemokine receptor, preferably selected from the group consisting of    CCR1, CCR2, CXCR4, and CXCR6, or at least one growth factor    receptor, preferably selected from the group consisting of    macrophage colony stimulating factor Receptor (CD115), granulocyte    colony stimulating factor Receptor (CD114), and    granulocyte-macrophage colony stimulating factor Receptor    (consisting of CD116 and CD131); monocytes of these characteristics    are particular suitable to treat inflammatory conditions and cancer;-   (iv) the lymphocyte is selected from the group consisting of a CD3⁺    and CD4⁺ or CD8⁺ T lymphocyte, or a CD19⁺, CD20⁺, CD21⁺, CD19⁺    CD20⁺, CD19⁺ CD21⁺, CD20⁺ CD21⁺, or CD19⁺ CD20⁺ CD21⁺ B lymphocyte;    or a natural killer (NK) cell, preferably the NK cell is selected    from the group consisting of CD56⁺ and without CD3 expression, or    CD16⁺ CD56⁺, CD56⁺ CD94⁺, CD56⁺ CD158a⁺, CD56⁺ CD158f⁺, CD56⁺CD314⁺,    CD56⁺CD335⁺ cell; or-   (v) the granulocyte is selected from the group consisting of a    neutrophil, preferably a CD66b⁺ neutrophil, an eosinophil and a    basophil, preferably a CD193⁺ eosinophil.

In a preferred embodiment of the targeted delivery system of the presentinvention the activated macrophage:

-   (i) is producible by in vitro incubation of a monocyte or macrophage    or their precursors with a factor capable of altering expression    markers on macrophages, preferably    -   (a) with at least one M1 inducer,    -   (b) with at least one M2 inducer,    -   (c) or with a factor capable of altering the macrophages ability        to secrete cytokines, preferably IL-10 and IL-12, chemokines        and/or to produce iNOS, arginase or other immunomodulating        enzymes; examples of such factors are: activated platelets,        IL-4, IL-10, IL-13, immune complex of an antigen and antibody,        IgG, heat activated gamma-globulin, glucocorticosteroid, tumour        growth factor-β (TGF-β), IL-1R, CC-chemokine ligand 2 (CCL-2),        IL-6, Macrophage colony-stimulating factor (M-CSF), peroxisome        proliferator-activated receptor γ (PPARγ) agonist, leukocyte        inhibitory factor (LIF), adenosine, helminth and fungal        infection, lipopolysaccharide (LPS), interferon γ (INF-γ), viral        and bacterial infection; in this respect it was observed that        activation of a monocyte with a M1 inducer, particularly LPS        will cause cell to express iNOS, that activation of a monocyte        with a M1 inducer, particularly LPS will cause cell not to        express Arginase-1, that activation of a monocyte with a M2        inducer, particularly IL-4 will cause cell to express        Arginase-1, and that activation of a monocyte with a M2 inducer,        particularly IL-4 will cause cell not to express iNOS,-   (ii) is characterized by expression of at least one of following    antigens: CD64, CD86, CD16, CD32, high expression of MCHII, and/or    production of iNOS and/or IL-12;-   (iii) is producible by in vitro incubation of a monocyte or    macrophage with a factor capable of inducing the ability of the    macrophage to phagocytose, e.g. IL-18, opsonins (for example    complement-derived proteins such as iC3b, immunoglobulin G),    calcitonin gene-related peptide (CGRP), lipopolysaccharide (LPS),    interferon γ (INF-γ), viral infection and/or bacterial infection;-   (iv) is characterized by expression of at least one of following    antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR),    CXC-motive chemokine receptor 4 (CXCR4), CD163, and/or T cell    immunoglobulin-domain and mucin-domain 2 (TIM-2), and/or show low    expression of MHCII; activated macrophages having these properties    are particularly suitable for complexes comprising ferritin as the    iron binding protein;-   (v) has the ability to phagocytose; and/or-   (vi) is capable of cytokine secretion, preferably of IL-12, or    IL-10, or production of inducible nitric oxide synthetase (iNOS) (or    other pro-inflammatory compounds), arginase or other    immunosuppressive/anti-inflammatory compounds.

In a preferred embodiment of the targeted delivery system of the presentinvention the M1 inducer for differentiating macrophages into M1macrophages is selected from the group consisting of lipopolysaccharide(LPS), interferon γ (INF-γ), and viral and bacterial infection and theM2 inducer for differentiating macrophages into M2 macrophages isselected from the group consisting of IL-4, IL-10, IL-13, immune complexof an antigen and antibody, IgG, heat activated gamma-globulin,glucocorticosteroid, tumour growth factor-0 (TGF-β), IL-1R, CC-chemokineligand 2 (CCL-2), IL-6, Macrophage colony-stimulating factor (M-CSF),peroxisome proliferator-activated receptor γ (PPARγ) agonist, leukocyteinhibitory factor (LIF), adenosine, helminth and fungal infection.

It has been surprisingly found by the present inventors that bothcomplex loaded M1 macrophages and M2 macrophages are suitable fortargeted active agent delivery into hypoxic tissues, preferably a tumouror its metastasis. In general we observed that 3 to 5% of theadministered M1 macrophages localized at the tumour site while about 35%of the M2 macrophages showed tumour specific targeting. However, thisgeneral advantage of M2 macrophages were offset when using a complexcomprising haemoglobin and drug, since significantly larger amounts ofthis complex could be loaded into M1 macrophages than into M2macrophages. Generally this specific tropisms makes M2 macrophages moresuitable for treating tumour and diseases characterized by hypoxictissue.

The present inventors also found CCL2-activated macrophages (bone marrowderived) are particularly suitable to target lymph nodes, preferablyinflamed lymph nodes. Thus, the use of such macrophages is preferred inall uses in which the targeting of lymph nodes is desired, in particularfor prophylactic or therapeutic vaccination as well as for diagnosis ofinflamed lymph nodes. Accordingly, the loading of CCL2-activatedmacrophages with complexes of iron binding proteins and antigens ispreferred.

In a preferred embodiment of the targeted delivery system of the presentinvention the monocyte-macrophage:

-   (i) is producible from a CD34⁺ hematopoietic precursor cell;-   (ii) is producible by in vitro incubation of monocytes with at least    one inducer, preferably M1 or M2 inducer, more preferably at least    one M2 inducer;-   (iii) is characterized by expression of at least one of the    following antigens: TfR, CD163, TIM-2, CD14, CD16, CD33, and/or    CD115;-   (iv) is characterized by expression of at least one of the following    antigens: TfR, CD163, TIM-2, CXCR4, CD14, and/or CD16; and/or-   (v) has the ability to phagocytose; and/or-   (vi) is not a dendritic cell which differentiation is controlled by    one or more of the following transcription factors: IRF8, NFIL3,    BATF3 or RELB, PU/1, RBPJ, IRF4 or TCF4.

In this embodiment of the targeted delivery system of the presentinvention the M1 inducer for differentiating monocyte-macrophage cellsis selected from the group consisting of LPS, INF-γ or viral orbacterial infection or the M2 inducer for differentiating monocytes isselected from the group consisting of IL-4, IL-10, IL-13, immune complexof an antigen and antibody, IgG, heat activated gamma-globulins,Glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonist,Leukocyte inhibitory factor (LIF), cancer-conditioned medium, cancercells, adenosine and helminth or fungal infection.

It has been surprisingly found by the present inventors that monocytesare suitable for targeted active agent delivery into hypoxic tissues,preferably the tumour or its metastasis while monocytes treated with M2activators are more suitable for targeted active agent delivery intohypoxic tissues, preferably the tumour or its metastasis. This specifictropisms make monocytes treated with M2 activators more suitable totargeting tumour and hypoxic sites, e.g. sites of inflammation.

In a preferred embodiment of the targeted delivery system of the presentinvention the lymphocyte:

-   (i) is obtainable from blood, spleen, or bone marrow or is    producible from a CD34⁺ precursor cell as known to the skilled    person and also described in the, e.g. Lefort and Kim, 2010, J Vis    Exp 40: 2017; Tassone and Fidler, 2012, Methods in Molecular Biology    882: 351-357; Kouro et al. 2005, Current Protocols in Immunology,    66:F22F.1:22F.1.1-22F.1.9;-   (ii) is an immunologically competent lymphocyte;-   (iii) expresses antigen specific T cell receptors; and/or-   (iv) is characterized by expression of at least one of the following    antigens: (a) CD3 and CD4 or CD8 or (b): CD19, CD20, CD21, CD19    CD20, CD19 CD21, CD20 CD21, or CD19 CD20 CD21 antigen, and is    preferably capable of producing immunoglobulins In a particularly    preferred embodiment the CD45+ lymphocytes is a NK cell, which-   (i) is obtainable from blood, spleen or bone marrow or producible    from a CD34⁺ precursor cell; and/or-   (ii) is characterized by the lack of CD3 expression and expression    of at least one of the following CD56⁺ and/or CD94⁺, CD158a⁺ CD158f₊    CD314⁺ CD335⁺.

In a preferred embodiment of the targeted delivery system of the presentinvention the granulocyte:

-   (i) is obtainable from blood, spleen or bone marrow or producible    from a CD34⁺ precursor cell as described, e.g. in Kuhs et al. 2015,    Curr Protoc Immunol 111:7.23-1-7.23.16; Coquery et al. 2012,    Cytometry A 81(9): 806-814; Swemydas and Lionakis 2013, J Vis Exp    77: 50586;-   (ii) is characterized by expression of at least one of the following    CD66b and/or CD193;-   (iii) is a polymorphonuclear leukocyte characterized by the presence    of granules in its cytoplasm; and/or-   (iii) is characterized by expression of at least one of the    following: TfR, CD163, TIM-2, and/or CXCR4.

In a preferred embodiment of the targeted delivery system of the presentinvention the iron binding protein is selected from the group consistingof ferritin, preferably heavy (H) type ferritin, light (L) ferritinand/or mitochondrial ferritin; haemoglobin, preferably haemoglobin A,haemoglobin AS, haemoglobin SC, haemoglobin C, haemoglobin D,haemoglobin E, haemoglobin F, haemoglobin H; haemoglobin-haptoglobincomplex, haemopexin, transferrin; and lactoferrin. The terms ferritin;haemoglobin, preferably haemoglobin A, haemoglobin AS, haemoglobin SC,haemoglobin C, haemoglobin D, haemoglobin E, haemoglobin F, haemoglobinH; haemoglobin-haptoglobin complex, hemopexin, transferrin; andlactoferrin encompass structural variants of the naturally occurringproteins and, thus relates to proteins that have at least 70%,preferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least 95%more preferably at least 100% of the ability of the respective wild-typeprotein to bind iron ion(s). The iron binding proteins used in thecontext of the present invention are preferably of mammalian, morepreferably mouse, rat, dog, ape, in particular, chimpanzee, or human,most preferably of human origin. Consensus sequences of the preferrediron binding proteins used in the context of the present invention areshown in FIG. 1 below. Preferred structural variants are based on thesequences indicated in FIG. 1. The residues marked with X vary amongdifferent mammalian ferritins, transferrins, and haemoglobins. Thealteration of these residues is not crucial for the ability of theproteins to bind to iron ions. Accordingly, it is preferred that aminoacid mutations or deletions effect one or more of the residues markedwith an X.

Plasma proteins have always been privileged carriers for the delivery ofactive pharrrra ingredients in cancer therapy. Thus, albumin, the mostabundant plasma protein, is currently used in therapeutic protocols forthe delivery of taxane molecules and doxorubicin derivatives (Larsen M Tet al. 2016, Mol Cell Ther 27; 4:3).

Human transferrin and ferritin proteins have been considered aseffective carriers for the delivery of small molecules ortoxin-conjugates to specifically target cancer cells. To date; in spiteof considerable efforts, no successful transferrin or ferritin drugcomplexes have however reached the clinic (Luck A N et al. 2013, AdvDrug Deliv Rev 65(8):1012-9).

Ferritin has a cage architecture and capability of iron-binding whichcould be used to encapsulate pharmaceutically active substances and/orlabels inside its cavity. Ferritins are not abundant in plasma, but canbe readily produced in high yield as recombinant proteins in commonprotein expression vectors such as Escherichia coli cells. Ferritins H-or L-chains are encoded as small protein monomer (21 kDa and 19 KDa forH and L chains, respectively) capable of a 24-mer assembly into acage-like structure, delimiting a 8 nm diameter cavity. The presentinventors noted in the context of working on the present invention thatH-ferritin homopolymers, represent a preferred protein construct inorder to specifically deliver encapsulated drugs to CD45⁺ leukocytecells expressing TfR. Furthermore H-ferritin targets complexpharmaceutically active substances, labels or pharmaceutically activesubstances and labels to the cell nucleus (and therefore directlydelivers DNA-binding proteins into the nucleus.

Purified transferrin can be efficiently conjugated to various moleculesincluding anticancer drugs through covalent linkers that areappropriately released inside the cells (Beyer U et al. 1998, J Med Chem41(15):2701-2708). In case of transferrin, only lysine groups on theprotein surface are ready available for covalent attachment.

Haemoglobin has been considered in the past as a possible drug carrier,due to its versatility in chemical conjugation with drugs, its abundanceand relative stability in the blood (Somatogen, 1993, WO1993008842 A1).Nevertheless, the lack of receptor targeting properties did not fosterbiomedical applications other than blood substitutes or antisicklingagent. As a matter of fact, Hb can only be recognized by CD163(haptoglobin/haemoglobin receptor) epitopes on the leukocytes,especially monocyte-macrophage origin. The CD45⁺ leukocyte, inparticular macrophage based protein delivery, described in thisapplication moved haemoglobin center stage as a target specific carrierof pharmaceutically active substances and/or labels. Haemoglobin can bereadily covalently linked to appropriate pharmaceutically activesubstances and/or labels, host hydrophobic pharmaceutically activesubstances and/or labels within the heme binding pocket or eventransport small molecules, e.g. cytotoxic molecules linked to the hemeiron. Hb can be easily modified by selective attachment of theappropriate drug conjugate to the beta93 cysteine residue, the onlytitratable cystein on the protein surface. Maleimido functionalizeddrugs, such as the tubuline inhibitor MonomethylAuristatin (MMAE) or theDNA crosslinking drug Pyrrolobenzodiazepine dimer (PBD) are most notableexamples of extremely potent cytotoxics that can be readily andspecifically attached to the relevant cys beta93 residue.

Alternatively, lysine residues on the Hb surface (at least 10 titratablelysine residues per Hb tetramer) may be easily amenable to drugconjugation through cleavable succinimide linkers. Haemoglobin alsooffers a unique capability of releasing non covalently bound heme groupat acidic pH values. Apo-haemoglobin thus obtained is capable of hostingseveral hydrophobic molecules within the empty heme pocket, as shown inthe case of paclitaxel (Meng Z et al. 2015 J Pharm Sci 104(3):1045-55)or for labels with fluorescent properties (e.g. chlorine e6, hyperycin,phtalocyanine derivatives) (Bong J et al. J Photochem Photobiol B 2014,140:163-172).

Whatever the conjugation/adsorption/binding method, haemoglobin (Fib),transferrin (Tf) and ferritins were shown by the present inventors to beprivileged carriers of pharmaceutically active substances and labels,once loaded into appropriate cell systems with tumour targetingproperties, e.g. activated macrophages. The easy, fast, cheap and safepurification procedure of these protein also provide a tremendous addedvalue.

Based on sequences of mammalian H-type ferritins, L-type ferritins,haemoglobin alpha chains, haemoglobin beta chains and transferrins aconsensus sequence was determined for each of these proteins. These areshown in FIG. 1 in SEQ ID NO: 2, 7, 9, 14, 16, 20, and 25,respectively). On this basis but also on the basis of deletion andstructural analysis disclosed in the prior art a minimal fragment wasdetermined for H-type ferritins, L-type ferritins, haemoglobin alphachains, and haemoglobin beta chains sufficient for uptake by CD45⁺leukocytes. These are shown in SEQ ID NO: 1, 3, 5 (H-type ferritin); 8,10, 12 (L-type ferritin), 15 and 17 (haemoglobin alpha chain) and 19 and21 (haemoglobin beta chain. Transferrin comprises a N-terminally locateddomain and a C-terminally located domain that are necessary for bindingiron and uptake by CD45⁺ leukocytes, if comprised in one polypeptide andpositioned between 100 to 450 amino acids apart, preferably between 150to 400, more preferably between 200 to 350 amino acids and morepreferably 250 to 320 amino acids apart. The N-terminal domain comprisesamino acids 1 to 82 of full length consensus transferrin (SEQ ID NO: 25)or full length human transferrin (SEQ ID NO: 28). The C-terminal domaincomprises amino acids 396 to 510 of full length consensus transferrin(SEQ ID NO: 25) or full length human transferrin (SEQ ID NO: 28). Ineach case an X is indicated in the consensus sequence it independentlystands for any amino acid and characterizes an amino acid not or onlypoorly conserved among mammalian H-type ferritins, which can be mutatedwithout or little detriment to the iron binding properties of therespective iron binding protein. It is preferred that X in each casetakes on the meaning of the amino acid of the respective human ironbinding protein aligning with X. This information can be taken, e.g.from FIG. 1, which shows alignments of the consensus sequences withhuman an in some instances mouse proteins.

Preferred H-type ferritins comprise or consist of the amino acidsequence indicated in SEQ ID NO: 1 and variants thereof having at least70% amino acid identity, more preferably at least 75% amino acididentity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a H-typeferritin consisting of the amino acid sequence according to SEQ ID NO: 1to be taken up by CD45⁺ leukocytes, preferably M2 leukocytes. Within SEQID NO: 1 X at position 5 may be present or absent, if present it meansany amino acid, preferably Ile, X at position 6 means any amino acid,preferably Asn, X at position 14 means any amino acid, preferably Tyr, Xat position 24 means any amino acid, preferably Tyr or Cys, X atposition 66 means any amino acid, preferably Phe, X at position 68 meansany amino acid, preferably Gln, X at position 75 means any amino acid,preferably Arg or Cys, X at position 90 means any amino acid, preferablyHis, X at position 94 means any amino acid, preferably Ser or Asn, X atposition 120 may be present or absent, if present it means any aminoacid, preferably His or Tyr, more preferably His, X at position 123means any amino acid, preferably Asn or Ser, more preferably Asn, X atposition 128 means any amino acid, preferably Ala or Ser, morepreferably Ala.

In a preferred embodiment the H-type ferritin comprises or consists ofmurine ferritin according to SEQ ID NO: 3. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a H-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 3 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the H-type ferritin comprises or consists ofhuman ferritin according to SEQ ID NO: 5. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a H-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 5 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the H-type ferritin comprises or consists of amammalian consensus sequence derived from aligning full length H-typeferritins according to SEQ ID NO: 2 or 7, with 2 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a H-type ferritin consisting of theamino acid sequence according to SEQ ID NO: 2 or 7, with 2 beingpreferred to be taken up by CD45⁺ leukocytes, preferably M2 macrophages.In SEQ ID NO: 2 X at position 6 can be any naturally occurring aminoacid, preferably Pro, X at position 14 can be any naturally occurringamino acid, preferably His, X at position 16 can be any naturallyoccurring amino acid, preferably Asp, X at position 21 may be present orabsent, if present it means any amino acid, preferably Ile, X atposition 22 means any amino acid, preferably Asn, X at position 30 canbe any naturally occurring amino acid, preferably Tyr, X at position 40can be any naturally occurring amino acid, preferably Tyr or Cys, morepreferably Tyr, X at position 82 can be any naturally occurring aminoacid, preferably Phe, X at position 84 can be any naturally occurringamino acid, preferably Gln, X at position 91 can be any naturallyoccurring amino acid, preferably Arg or Cys, more preferably Cys, X atposition 106 can be any naturally occurring amino acid, preferably His,X at position 110 can be any naturally occurring amino acid, preferablyAsn or Ser, more preferably Asn, X at position 137 can be any naturallyoccurring amino acid, preferably His or Tyr, more preferably His, X atposition 140 can be any naturally occurring amino acid, preferably Asnor Ser, more preferably Asn, X at position 145 can be any naturallyoccurring amino acid, preferably Ala or Ser, more preferably Ala, X atposition 164 can be any naturally occurring amino acid, preferably Alaor Ser, more preferably Ser, X at position 166 can be any naturallyoccurring amino acid, preferably Met or Leu, preferably Leu, X atposition 178 can be any naturally occurring amino acid, preferably Aspor His, more preferably Asp, X at position 181 is absent or anynaturally occurring amino acid, preferably Asn, X at position 182 isabsent or any naturally occurring amino acid, preferably Glu, X atposition 183 is absent or any naturally occurring amino acid, preferablySer. In SEQ ID NO: 7 X at position 6 can be any naturally occurringamino acid, preferably Pro X at position 14 can be any naturallyoccurring amino acid, preferably His, X at position 16 can be anynaturally occurring amino acid, preferably Asp, X at position 21 may bepresent or absent, if present it means any amino acid, preferably Ile, Xat position 29 can be any naturally occurring amino acid, preferablyTyr, X at position 81 can be any naturally occurring amino acid,preferably Phe, X at position 83 can be any naturally occurring aminoacid, preferably Gln, X at position 105 can be any naturally occurringamino acid, preferably His, X at position 144 can be any naturallyoccurring amino acid, preferably Ala or Ser, more preferably Ala, X atposition 180 is absent or any naturally occurring amino acid, preferablyAsn, X at position 181 is absent or any naturally occurring amino acid,preferably Glu, X at position 182 is absent or any naturally occurringamino acid, preferably Ser.

In a preferred embodiment the H-type ferritin comprises or consists ofmurine full length ferritin according to SEQ ID NO: 4 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a murine H-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 4 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the H-type ferritin comprises or consists ofhuman full length ferritin according to SEQ ID NO: 6 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human H-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 6 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

Preferred L-type ferritins comprise or consist of the amino acidsequence indicated in SEQ ID NO: 8 and variants thereof having at least70% amino acid identity, more preferably at least 75% amino acididentity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a L-typeferritin consisting of the amino acid sequence according to SEQ ID NO: 8to be taken up by CD45⁺ leukocytes, preferably M2 leukocytes. In SEQ IDNO: 8 X at position at position 5 can be any naturally occurring aminoacid, preferably Asp or Glu, more preferably Asp, X at position 12 canbe any naturally occurring amino acid, preferably Arg or Ser, morepreferably Ser, X at position 17 can be any naturally occurring aminoacid, preferably Ser or Arg, more preferably Ser, X at position 19 canbe any naturally occurring amino acid, preferably Arg or Gln, morepreferably Gln, X at position 29 can be any naturally occurring aminoacid, preferably Phe, X at position 30 can be any naturally occurringamino acid, preferably Tyr or Phe, more preferably Tyr, X at position 42can be any naturally occurring amino acid, preferably Ser or Gly, morepreferably Ser, X at position 56 can be any naturally occurring aminoacid, preferably Ala or Tyr, more preferably Tyr, X at position 61 canbe any naturally occurring amino acid, preferably Glu or Lys, morepreferably Lys, X at position 62 can be any naturally occurring aminoacid, preferably Met or Phe, more preferably Met, X at position 65 canbe any naturally occurring amino acid, preferably Asp or Gln, morepreferably Gln, X at position 75 can be any naturally occurring aminoacid, preferably Ile or Val, more preferably Ile, X at position 76 canbe any naturally occurring amino acid, preferably Lys or Gln, morepreferably Lys, X at position 79 can be any naturally occurring aminoacid, preferably Ala or Ser, more preferably Ala, X at position 80 canbe any naturally occurring amino acid, preferably Glu or Gln, morepreferably Gln, X at position 87 can be any naturally occurring aminoacid, preferably Pro or Gln, more preferably Pro, X at position 88 canbe any naturally occurring amino acid, preferably Glu or Asp, morepreferably Asp, X at position 91 can be any naturally occurring aminoacid, preferably Glu or Lys, more preferably Lys, X at position 94 canbe any naturally occurring amino acid, preferably Met or Leu, morepreferably Leu, X at position 96 can be any naturally occurring aminoacid, preferably Met or Leu, more preferably Met, X at position 99 canbe any naturally occurring amino acid, preferably Lys or Asn, preferablyLys, X at position 115 can be any naturally occurring amino acid,preferably Thr or Ala, more preferably Thr, X at position 119 can be anynaturally occurring amino acid, preferably Leu, X at position 125 can beany naturally occurring amino acid, preferably Ser or Thr, morepreferably Thr, X at position 127 can be any naturally occurring aminoacid, preferably Tyr or Phe, more preferably Phe, X at position 130 canbe any naturally occurring amino acid, preferably Lys or Glu, morepreferably Glu, X at position 140 can be any naturally occurring aminoacid, preferably Asp or Asn, more preferably Asp, X at position 146 canbe any naturally occurring amino acid, preferably Arg or His, morepreferably His, and X at position 148 can be any naturally occurringamino acid, preferably Leu or Val, more preferably Leu.

In a preferred embodiment the L-type ferritin comprises or consists ofmurine L-type ferritin according to SEQ ID NO: 10. Accordingly,preferred structural variants have at least 70% amino acid identity,more preferably at least 75% amino acid identity, more preferably atleast 80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a L-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 10 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the L-type ferritin comprises or consists ofhuman ferritin according to SEQ ID NO: 12. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a L-type ferritin consisting of the amino acidsequence according to SEQ ID NO: 12 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the L-type ferritin comprises or consists of amammalian consensus sequence derived from aligning full length H-typeferritins according to SEQ ID NO: 9 or 14, with 9 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a L-type ferritin consisting of theamino acid sequence according to SEQ ID NO: 9 or 14, with 9 beingpreferred to be taken up by CD45⁺ leukocytes, preferably M2 macrophages.In SEQ ID NO: 9 X at position 12 X at position 12 can be any naturallyoccurring amino acid, preferably Asp or Glu, more preferably Asp, X atposition 19 can be any naturally occurring amino acid, preferably Ser orArg, more preferably Ser, X at position 24 can be any naturallyoccurring amino acid, preferably Ser or Arg, more preferably Ser, X atposition 26 can be any naturally occurring amino acid, preferably Arg orGln, more preferably Gln, X at position 36 can be any naturallyoccurring amino acid, preferably Phe, X at position 37 can be anynaturally occurring amino acid, preferably Tyr or Phe, more preferablyTyr, X at position 47 can be any naturally occurring amino acid,preferably Ser or Gly, more preferably Ser, X at position 63 can be anynaturally occurring amino acid, preferably Ala or Tyr, more preferablyTyr, X at position 68 can be any naturally occurring amino acid,preferably Glu or Lys, more preferably Lys, X at position 69 can be anynaturally occurring amino acid, preferably Met or Phe, more preferablyMet, X at position 72 can be any naturally occurring amino acid,preferably Asp or Gln, more preferably Gln, X at position 82 can be anynaturally occurring amino acid, preferably Ile or Val, more preferablyIle, X at position 83 can be any naturally occurring amino acid,preferably Lys or Gln, more preferably Lys, X at position 86 can be anynaturally occurring amino acid, preferably Ala or Ser, more preferablyAla, X at position 87 can be any naturally occurring amino acid,preferably Glu or Gln, more preferably Gln, X at position 94 can be anynaturally occurring amino acid, preferably Pro or Gln, more preferablyPro, X at position 95 can be any naturally occurring amino acid,preferably Glu or Asp, more preferably Asp, X at position 98 can be anynaturally occurring amino acid, preferably Glu or Lys, more preferablyLys, X at position 101 can be any naturally occurring amino acid,preferably Met or Leu, more preferably Leu, X at position 103 can be anynaturally occurring amino acid, preferably Met or Leu, more preferablyMet, X at position 106 can be any naturally occurring amino acid,preferably Lys or Asn, preferably Lys, X can be any naturally occurringamino acid, X at position 126 can be any naturally occurring amino acid,preferably Leu, X at position 132 can be any naturally occurring aminoacid, preferably Ser or Thr, more preferably Thr, X at position 134 canbe any naturally occurring amino acid, preferably Tyr or Phe, morepreferably Phe, X at position 137 can be any naturally occurring aminoacid, preferably Lys or Glu, more preferably Glu, X at position 147 canbe any naturally occurring amino acid, preferably Asp or Asn, morepreferably Asp, X at position 153 can be any naturally occurring aminoacid, preferably Arg or His, more preferably His, X at position 155 canbe any naturally occurring amino acid, preferably Leu or Val, morepreferably Leu, X at position 161 can be absent or any naturallyoccurring amino acid, preferably Ala, X at position 163 can be absent orany naturally occurring amino acid, preferably Thr, X at position 166can be absent or any naturally occurring amino acid, preferably Pro, andX at position 168 can be any naturally occurring amino acid, preferablyGly or Ala, more preferably Ala. In SEQ ID NO: 14 X at position 36 canbe any naturally occurring amino acid, preferably Phe, X at position 37can be any naturally occurring amino acid, preferably Tyr or Phe, morepreferably Tyr, X at position 94 can be any naturally occurring aminoacid, preferably Pro or Gln, more preferably Pro, X at position 126 canbe any naturally occurring amino acid, preferably Leu, X at position 137can be any naturally occurring amino acid, preferably Lys or Glu, morepreferably Glu, X at position 147 can be any naturally occurring aminoacid, preferably Asp or Asn, more preferably Asp, X can be any naturallyoccurring amino acid, X at position 163 can be absent or any naturallyoccurring amino acid, preferably Thr, X at position 166 can be absent orany naturally occurring amino acid, preferably Pro, X at position 168can be any naturally occurring amino acid, preferably Gly or Ala, morepreferably Ala.

In a preferred embodiment the L-type ferritin comprises or consists ofmurine full length ferritin according to SEQ ID NO: 11 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a murine L-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 11 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the L-type ferritin comprises or consists ofhuman full length ferritin according to SEQ ID NO: 13 being preferred.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human L-type ferritin consistingof the amino acid sequence according to SEQ ID NO: 13 to be taken up byCD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal mammalian consensus sequence derived from aligning full lengthalpha haemoglobins according to SEQ ID NO: 15 Preferred comprise orconsist of the amino acid sequence indicated in SEQ ID NO: 15 andvariants thereof having at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of an alpha haemoglobin consisting of the amino acidsequence according to SEQ ID NO: 15 to be taken up by CD45⁺ leukocytes,preferably M1 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal human amino acid sequence derived of human alpha haemoglobinaccording to SEQ ID NO: 17. Accordingly, preferred structural variantshave at least 70% amino acid identity, more preferably at least 75%amino acid identity, more preferably at least 80% amino acid identity,more preferably at least 85% amino acid identity, more preferably atleast 90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of an alphahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 17 to be taken up by CD45⁺ leukocytes, preferably M1 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa mammalian consensus sequence derived from aligning full length alphahaemoglobins according to SEQ ID NO: 16. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of an alpha haemoglobins consisting of the amino acidsequence according to SEQ ID NO: 16 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofhuman full length alpha haemoglobin according to SEQ ID NO: 18.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human full length alphahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 18 to be taken up by CD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal mammalian consensus sequence derived from aligning full lengthbeta haemoglobins according to SEQ ID NO: 19 and variants thereof havingat least 70% amino acid identity, more preferably at least 75% aminoacid identity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a betahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 19 to be taken up by CD45⁺ leukocytes, preferably M1 macrophages.

In a preferred embodiment the alpha haemoglobin comprises or consists ofa minimal human amino acid sequence derived of human beta haemoglobinaccording to SEQ ID NO: 21. Accordingly, preferred structural variantshave at least 70% amino acid identity, more preferably at least 75%amino acid identity, more preferably at least 80% amino acid identity,more preferably at least 85% amino acid identity, more preferably atleast 90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a betahaemoglobin consisting of the amino acid sequence according to SEQ IDNO: 21 to be taken up by CD45⁺ leukocytes, preferably M1 macrophages.

In a preferred embodiment the beta haemoglobin comprises or consists ofa mammalian consensus sequence derived from aligning full length betahaemoglobins according to SEQ ID NO: 20. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of an beta haemoglobins consisting of the amino acidsequence according to SEQ ID NO: 20 to be taken up by CD45⁺ leukocytes,preferably M2 macrophages.

In a preferred embodiment the beta haemoglobin comprises or consists ofhuman full length beta haemoglobin according to SEQ ID NO: 22.Accordingly, preferred structural variants have at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a human full length beta haemoglobinconsisting of the amino acid sequence according to SEQ ID NO: 22 to betaken up by CD45⁺ leukocytes, preferably M2 macrophages.

In a preferred embodiment the transferrin comprises or consists of amammalian consensus sequence derived from aligning full length alphahaemoglobins according to SEQ ID NO: 25. Thus, particularly, preferredtransferrins comprise or consist of the amino acid sequence indicated inSEQ ID NO: 25 and of variants thereof having at least 70% amino acididentity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a transferrin consisting of theamino acid sequence according to SEQ ID NO: 25 to be taken up by CD45⁺leukocytes preferably M1 macrophages.

In a preferred embodiment the transferrin comprises or consists of humantransferrin according to SEQ ID NO: 28. Accordingly, preferredstructural variants have at least 70% amino acid identity, morepreferably at least 75% amino acid identity, more preferably at least80% amino acid identity, more preferably at least 85% amino acididentity, more preferably at least 90% amino acid identity, morepreferably at least 95% amino acid identity and in each case at least70% of the ability of a transferrin consisting of the amino acidsequence according to SEQ ID NO: 28 to be taken up by CD45⁺ leukocytes,preferably M1 macrophages.

The iron binding properties of transferrins are dependent on aN-terminally and C-terminally located domain. Thus, in a preferredembodiment the transferrin used in the present invention comprises atleast the N-terminal domain according to SEQ ID NO: 23 and theC-terminal domain according to SEQ ID NO: 24. Preferred transferrincomprise proteins that comprise the amino acid sequence indicated in SEQID NO: 23 and 24 as well as variants thereof having at least 70% aminoacid identity, more preferably at least 75% amino acid identity, morepreferably at least 80% amino acid identity, more preferably at least85% amino acid identity, more preferably at least 90% amino acididentity, more preferably at least 95% amino acid identity and in eachcase at least 70% of the ability of a transferrin consisting of theamino acid sequence according to SEQ ID NO: 23 and 24 to be taken up byCD45⁺ leukocytes, preferably M1 macrophages. SEQ ID NO: 23 or 24indicates a consensus sequence of mammalian transferrins.

Thus, in a preferred embodiment the transferrin used in the presentinvention comprises at least the N-terminal domain according to SEQ IDNO: 26 and the C-terminal domain according to SEQ ID NO: 27. Preferredtransferrin comprise proteins that comprise the amino acid sequenceindicated in SEQ ID NO: 26 and 27 as well as variants thereof having atleast 70% amino acid identity, more preferably at least 75% amino acididentity, more preferably at least 80% amino acid identity, morepreferably at least 85% amino acid identity, more preferably at least90% amino acid identity, more preferably at least 95% amino acididentity and in each case at least 70% of the ability of a transferrinconsisting of the amino acid sequence according to SEQ ID NO: 26 and 27,respectively, to be taken up by CD45⁺ leukocytes, preferably M1macrophages.

Preferred ferritins, also comprise proteins that, irrespective of thegiven amino acid sequence, conform to the 24-mer subunit assembly of afour helix bundle protein module, falling within given sequencealignments of distantly related proteins as defined by 3D structurebased alignments.

It is a surprising observation of the present inventors that monocytes,macrophages and preferably M2 macrophages are able to uptake the amountof label enough to make them visible using imaging methods at the siteof their accumulation (e.g. in the tumour or its metastasis, hypoxiasite). Surprisingly, inventors found out that lymphocytes and M2macrophages are better in uptake of complexes comprising one or moreferritin and one or more pharmaceutically active substance, label orpharmaceutically active substance and label, that M1 macrophages arebetter in uptake of complexes comprising one or more haemoglobin and oneor more pharmaceutically active substance, label or pharmaceuticallyactive substance and label and that macrophages are better in uptake ofcomplexes comprising one or more transferrin and one or morepharmaceutically active substance, label or pharmaceutically activesubstance and label. Accordingly, based on the tissue and cellulartropism of CD45⁺ leukocytes: monocytes, M1 and M2 macrophages,granulocytes and lymphocytes, described above complexes comprising oneor more ferritin and one or more pharmaceutically active substance,label or pharmaceutically active substance and label are used to load M2macrophages, lymphocytes or monocytes if the tropism of M2 macrophages,lymphocytes or monocytes is desired and complexes comprising one or morehaemoglobin and one or more pharmaceutically active substance, label orpharmaceutically active substance and label are used to load M1macrophages, if the tropism of M1 macrophages is desired.

In a preferred embodiment of the targeted delivery system of the presentinvention the pharmaceutically active substance, label orpharmaceutically active substance and label is selected from the groupconsisting of a protein, a nucleic acid, a chemical non-proteinnon-nucleic acid compound with a molecular weight of less than 1.5 kD,more preferably less than 1 kD, preferably an anticancer drug, inparticular a cytostatic drug, cytotoxic drug and prodrugs thereof; ananti arteriosclerotic drug; and anti-inflammatory drug; andphotosensitizing compound; a virus, in particular oncolytic virus; and αa or ß radiation emitting radioisotope, which also emit a cell damagingamount of γ radiation, preferably selected from the group consisting oflutetium-177, ytterbium-90, iodine-131, samarium-153, phosphorus-32,caesium-131, palladium-103, radium-233, iodine-125, and boron-10 or acell damaging amount of a radiation, preferably selected from the groupconsisting of actinium-225, bismuth-213, lead-212, and polonium-212.Also preferred is a complex of above mentioned compounds and isotopeslinked to the nanoparticles (e.g. gold, argentum, graphen) or thesenanoparticles.

Preferred anticancer drugs are selected from an apoptosis/autophagy ornecrosis-inducing drug. An apoptosis/autophagy or necrosis-inducing drugcan be any drug that is able to induce apoptosis/autophagy or necrosiseffectively even in cells having an abnormality in cell proliferation.These drugs are preferably used in complexes with one or more ferritins.

Preferred anticancer drugs are selected from the group consisting of anapoptosis-inducing drug, an alkylating substance, anti-metabolites,antibiotics, epothilones, nuclear receptor agonists and antagonists, ananti-androgene, an anti-estrogen, a platinum compound, a hormone, aantihormone, an interferon, an inhibitor of cell cycle-dependent proteinkinases (CDKs), an inhibitor of cyclooxygenases and/or lipoxygenases, abiogeneic fatty acid, a biogenic fatty acid derivative, includingprostanoids and leukotrienes, an inhibitor of protein kinases, aninhibitor of protein phosphatases, an inhibitor of lipid kinases, aplatinum coordination complex, an ethyleneimine, a methylmelamine, atriazine, a vinca alkaloid, a pyrimidine analog, a purine analog, analkylsulfonate, a folic acid analog, an anthracendione, a substitutedurea, and a methylhydrazin derivative, an ene-diyne antibiotic, atubulin polymerization inhibitor such as a maytansinoid or anauristatine derivate, immune check-point inhibitor, and an inhibitor oftumour-specific protein or marker, preferably a Rho-GDP-dissociationinhibitor, more preferably Grp94, or AXL inhibitor.

Other preferred anticancer drugs are selected from the group consistingof acediasulfone, aclarubicine, ambazone, aminoglutethimide,L-asparaginase, azathioprine, banoxantrone, bendamustine, bleomycin,busulfan, calcium folinate, carboplatin, carpecitabine, carmustine,celecoxib, chlorambucil, cis-platin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin dapsone, daunorubicin,dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin,enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate,estrogen, ethinylestradiole, etoposide, flavopiridol, floxuridine,fludarabine, fluorouracil, fluoxymesterone, flutamide fosfestrol,furazolidone, gemcitabine, gonadotropin releasing hormone analog,hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin,hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine,ifosfamide, interferon α, irinotecan, leuprolide, lomustine, lurtotecan,mafenide sulfate olamide, mechlorethamine, medroxyprogesterone acetate,megastrolacetate, melphalan, mepacrine, mercaptopurine, methotrexate,metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone,mithramycin, nalidixic acid, nifuratel, nifuroxazide, nifuralazine,nifurtimox, nimustine, ninorazole, nitrofurantoin, nitrogen mustards,oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine,phthalylsulfathiazole, pipobroman, prednimustine, prednisone, preussin,procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib,rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustinestreptozocine, sulfacarbamide, sulfacetamide, sulfachlopyridazine,sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole,sulfafurazole, sulfaguanidine, sulfaguanole, sulfamethizole,sulfamethoxazole, co-trimoxazole, sulfamethoxydiazine,sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin,sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen,taxol, teniposide, tertiposide, testolactone, testosteronpropionate,thioguanine, thiotepa, tinidazole, topotecan, triaziquone, treosulfan,trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine,vinblastine, vinorelbine, and zorubicin, preferably selected from thegroup consisting of auristatin, banoxantrone, bendamustine,chlorambucil, chaliceamycin, cyclophosphamide dynemycin A, maytansine,melphalan, mertansine, and neocazinostatin, most preferablybanoxantrone, bendamustine, chlorambucil, cyclophosphamide,pyrrolobenzodiazepine and melphalan.

It is particularly preferred that the anticancer drug is a proliferationinhibiting protein, preferably a cell cycle inhibitor or an antibody orantibody like binding protein that specifically binds to a proliferationpromoting protein or a nucleic acid, preferably encoding a proliferationinhibiting protein or an antibody or antibody like binding protein thatspecifically binds to a proliferation promoting protein or a siRNA orDNAzym.

Preferred immunomodulatory drugs that activate or inhibit activity ofimmune cells. These can be natural or synthetic ligands or antagonistsof Pattern Recognition Receptors, particularly Toll-like Receptors,NOD-like receptors (NLR), RIG-I-like receptors (RLR). Physiologically,these receptors recognize class of signals known as pathogen-associatedmolecular patterns (PAMPs) and damage-associated molecular patterns(DAMPs).

Preferred examples of antibodies to be used in the context of thepresent invention are single chain antibodies, antibody fragments,nanobodies, light or heavy chains, variable light or variable heavychains, or diabodies. Preferred antibody fragments comprise a fragmentantigen binding (Fab) fragment, a Fab′ fragment, a F(ab′)2 fragment, aheavy chain antibody, a single-domain antibody (sdAb), a single-chainfragment variable (scFv), a fragment variable (Fv), a VH domain, a VLdomain, a single domain antibody, a nanobody, an IgNAR (immunoglobulinnew antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), adual affinity re-targeting (DART) molecule, a triple body, a diabody, asingle-chain diabody, and a fusion protein thereof.

It has been surprisingly found by the present inventors that inflamedjoints are targeted by macrophage-monocytes, preferably activatedmacrophages. Thus, in diagnostic applications in which inflamed jointsare to be detected macrophage-monocytes are preferably loaded with alabel or a complex comprising an iron binding protein and a label. Intherapeutic applications, it is preferred that the complex comprises apharmaceutically active substance with anti-inflammatory activity todeliver the anti-inflammatory substance to the inflamed tissue.

It has been surprisingly found by the present inventors that thetargeted delivery system of the present invention targets lymph nodes,which makes it particularly suitable for delivery of antigens todendritic cells residing in the lymph nodes. The lymph node targeting isparticularly pronounced, if the cells loaded with the complex aremacrophages in particular activated macrophages, even more preferablyCCL-2 activated bone marrow derived activated macrophages, orlymphocytes, in particular B cells or T cells. Thus in a preferredembodiment the targeted delivery system is used to deliver one or moreantigens in order to elicit a prophylactic and/or therapeutic immuneresponse against the one or more antigens. Preferred antigens arederived from pathogens, i.e. bacteria or viruses or are tumour specificantigens. The term “tumour specific antigens” refers to proteins orepitopes (including peptides with altered glycosylation patterns) thatare higher expressed on tumour cells in comparison to non-tumour cells,preferably to antigens or epitopes only expressed on tumour cells.Preferred antigens are selected from the group consisting of epidermalgrowth factor receptor (EGFR, ErbB-1, HER1), ErbB-2 (HER2/neu),ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-like growth factorreceptor (IGFR) family, IGF-binding proteins (IGFBPs), IGFR ligandfamily; platelet derived growth factor receptor (PDGFR) family, PDGFRligand family; fibroblast growth factor receptor (FGFR) family, FGFRligand family, vascular endothelial growth factor receptor (VEGFR)family, VEGF family; HGF receptor family; TRK receptor family; ephrin(EPH) receptor family; AXL receptor family; leukocyte tyrosine kinase(LTK) receptor family; TIE receptor family, angiopoietin 1,2; receptortyrosine kinase-like orphan receptor (ROR) receptor family; discoidindomain receptor (DDR) family; RET receptor family; KLG receptor family;RYK receptor family; MuSK receptor family; Transforming growth factor α(TGF-α) receptors, TGF-β; Cytokine receptors, Class I (hematopoietinfamily) and Class II (interferon/IL-10 family) receptors, tumor necrosisfactor (TNF) receptor superfamily (TNFRSF), death receptor family;cancertestis (CT) antigens, lineage-specific antigens, differentiationantigens, alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson(Bcr-abl) fusion products, B-RAF, caspase-5 (CASP-5), caspase-8(CASP-8), β-catenin (CTNNB1), cell division cycle 27 (CDC27),cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-1, dek-can fusion protein,EFTUD-2, Elongation factor 2 (ELF2), Ets variant gene 6/acute myeloidleukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), GPNMB,low density lipid receptor/GDP-L fucose: β-Dgalactose2-α-Lfucosyltransferase (LDLR/FUT) fusion protein, HLA-A2. arginine toisoleucine exchange at residue 170 of the α-helix of the α2-domain inthe HLA-A2 gene (HLA-A*201-R170I), HLA-A11, heat shock protein 70-2mutated (HSP70-2M), KIAA0205, MART2, melanoma ubiquitous mutated 1, 2, 3(MUM-1, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin classI, NFYC, OGT, OS-9, pml-RARalpha fusion protein, PRDX5, PTPRK, K-ras(KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2fusion protein, Triosephosphate Isomerase, BAGE, BAGE-1, BAGE-2,3,4,5,GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrant N-acetyl glucosaminyl transferaseV, MGAT5), HERV-K-MEL, KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognizedantigen on melanoma (CAMEL), MAGE-A1 (MAGE-1), MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11,MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2,mucin 1 (MUC1), MART-1/Melan-A (MLANA), gp100, gp100/Pme117 (SILV),tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1, NY-ESO-1/LAGE-2, SAGE,Sp17, SSX-1,2,3,4, TRP2-INT2, carcino-embryonic antigen (CEA),Kallikrein 4, mammaglobin-A, OA1, prostate specific antigen (PSA),TRP-1/gp75, TRP-2, adipophilin, interferon inducible protein absent inmelanoma 2 (AIM-2), BING-4, CPSF, cyclin D1, epithelial cell adhesionmolecule (EpCAM), EphA3, fibroblast growth factor-5 (FGF-5),glycoprotein 250 (gp250), EGFR (ERBB1), HER-2/neu (ERBB2), interleukin13 receptor α2 chain (IL13Ralpha2), IL-6 receptor, intestinal carboxylesterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUC1, p53(TP53), PBF, PRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, survivin(BIRC5), human telomerase reverse transcriptase (hTERT), telomerase,Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1,CTAGE-1, CSAGE, MMA1, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC,MORC, SGY-1, SPO11, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE,TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors(ER), androgen receptors (AR), CD40, CD30, CD20, CD19, CD33, cancerantigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA19-9), β-human chorionic gonadotropin, 1-2 microglobulin, squamous cellcarcinoma antigen, neuron-specific enolase, heat shock protein gp96,GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinomaantigen recognized by T cells 4 (ART-4), carcinoembryogenic antigenpeptide-1 (CAP-1), calcium-activated chloride channel-2 (CLCA2),cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), Humanpapilloma virus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsidantigens, others), Epstein-Barr virus (EBV) proteins (EBV latentmembrane proteins-LMPL LMP2; others), Hepatitis B or C virus proteins,and HIV proteins.

If the pharmaceutically active substance is a nucleic acid it ispreferred that it is a miRNA, siRNA, chemically modified-RNA, DNAzyme ora nucleic acid encoding a pharmaceutically active protein, e.g. anantibody, an antibody mimetic, a cytokine, a prodrug-converting enzyme,an immunogenic peptide or the like.

In a preferred embodiment of the targeted delivery system of the presentinvention the label is selected from a fluorescent dye, a fluorescenceemitting isotope, a radioisotope, a detectable polypeptide or nucleicacid encoding such a detectable polypeptide and a contrast agent.

In a preferred embodiment of the targeted delivery system of the presentinvention the label comprises a chelating agent which forms a complexwith divalent or trivalent metal cations.

In a preferred embodiment of the targeted delivery system of the presentinvention the chelating agent is selected from the group consisting of1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid (DOTA),ethylenediaminetetraacetic acid (EDTA),1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),triethylenetetramine (TETA), iminodiacetic acid,Diethylenetriamine-N,N,N′,N′,N″-pentaacetic acid (DTPA) and6-Hydrazinopyridine-3-carboxylic acid (HYNIC).

In a preferred embodiment of the targeted delivery system of the presentinvention the contrast agent comprises a paramagnetic agent, preferablyselected from Gd, Eu, W and Mn, or ferrihydride.

In a preferred embodiment of the targeted delivery system of the presentinvention the radioisotope/fluorescence emitting isotope is selectedfrom the group consisting of alpha radiation emitting isotopes, gammaradiation emitting isotopes, Auger electron emitting isotopes, X-rayemitting isotopes, fluorescence emitting isotopes, such as ¹⁸F, ⁵¹Cr,fluorescent ⁶⁵Tb, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ^(99m)Tc, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁵³Sm,¹⁶⁶Ho, ⁸⁸Y, ⁹⁰Y, ¹⁴⁹Pm, ¹⁷⁷Lu, ⁴⁷Sc, ¹⁴²Pr, ¹⁵⁹Gd, ²¹²Bi, ⁷²As, ⁷²Se,⁹⁷Ru, ¹⁰⁹Pd, ¹⁰⁵Rh, ^(101m15)Rh, ¹¹⁹Sb, ¹²⁸Ba, ¹²³I, ¹²⁴I, ¹³¹I, ¹⁹⁷Hg,²¹¹At, ¹⁶⁹Eu, ²⁰³Pb, ²¹²Pb, ⁶⁴Cu, ⁸⁹Zr, ⁶⁷Cu, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁹⁸Au and¹⁹⁹Ag as well as conjugates and combinations of above with proteins,peptides, small molecular inhibitors, antibodies or other compounds(e.g. ¹⁸F-FDG, ⁸⁹Zr-oxide or ⁶⁴Cu-porfirin).

In a preferred embodiment of the targeted delivery system of the presentinvention the fluorescence dye is selected from the group consisting ofthe following classes of fluorescent dyes: Xanthens, Acridines,Oxazines, Cynines, Styryl dyes, Coumarines, Porphines,Metal-Ligand-Complexes, Fluorescent proteins, Nanocrystals, Perylenesand Phtalocyanines as well as conjugates and combinations of theseclasses of dyes.

In a preferred embodiment of the targeted delivery system of the presentinvention the detectable polypeptide is an autofluorescent protein,preferably green fluorescent protein or any structural variant thereofwith an altered adsorption and/or emission spectrum.

As has been outlined above, the targeted delivery system of the presentinvention has particular suitability to deliver pharmaceutically activesubstances, labels or pharmaceutically active substances and labels tohypoxic areas. Hypoxy is characteristic of various disease includingcancer and inflammatory diseases and thus allows targeting suchdiseases.

In addition to the targeting the use of pharmaceutically activesubstances, which are activated under hypoxic conditions adds a furtherspecificity to the targeting and/or further reduces adverse effects ofthe pharmaceutically active substances, labels or pharmaceuticallyactive substances and labels. Thus, in particularly preferredembodiments the pharmaceutically active substance, label orpharmaceutically active substance and label is a hypoxia-activatedprodrug. The backbone of all the hypoxia-activated prodrugs is thepresence of one of five different chemical moieties (nitro groups,quinines, aromatic and aliphatic N-oxides and transition metals) thatare enzymatically reduced under hypoxic conditions in tissue.Hypoxia-activated prodrugs are any prodrug that is less active orinactive, relative to the corresponding drug, and comprises the drug andone or more bioreducible groups. Such hypoxia-activated prodrugs includeall prodrugs activated by a variety of reducing agents and reducingenzymes, including without limitation single electron transferringenzymes (such as cytochrome P450 reductases) and two electrontransferring (or hydride transferring) enzymes. According to preferredembodiment of the invention hypoxia-activated prodrug is TH-302. Methodsof synthesizing TH-302 are described in PCT application WO 07/002931 andWO 08/083101. Preferably examples of such prodrugs are selected from theclass I group consisting of: benzotriazine N-oxides, apaziquone (E09),tirapazamine (TPN) and SN30000; or class II group consisting of: nitrocompounds PR104A, TH-302, TH-4000, and AQ4N.

In a preferred embodiment of the isolated targeted delivery system ofthe present invention the bond(s) between the iron binding protein(s)and the pharmaceutically active substance, label or pharmaceuticallyactive substance and label comprised in the complex are covalent and/ornon-covalent; and/or the pharmaceutically active substance, label orpharmaceutically active substance and label comprised in the complex isentrapped/encapsulated by the iron binding protein, preferably ferritinor multimers thereof. In one embodiment the covalent and/or non-covalentcoupling is indirect through a linker or spacer. If the formation ofcovalent bonds is desired, relevant thiol, amino or carboxyl groups ofthe iron binding proteins are used to covalently couple pharmaceuticallyactive substances, labels or pharmaceutically active substances andlabels directly or indirectly to the one or more iron binding protein.

It is also envisioned that different pharmaceutically active substances,labels or pharmaceutically active substances and labels are comprised inthe complex. For example, one type of pharmaceutically active substance,label or pharmaceutically active substance and label may be bound to aniron binding protein (non-covalently bound), while another type isencapsulated. This approach utilizes different release rates of thepharmaceutically active substances, labels or pharmaceutically activesubstances and labels from the iron binding protein once delivered tothe targeted tissue and/or cells. For example, pharmaceutically activesubstance, label or pharmaceutically active substance and label can becovalently attached to ferritin molecule either on the surface of the24-mer or within the internal cavity by exploiting the reactivity ofrelevant thiol, amino or carboxyl groups. The types of such usefulreactions are well known in the art and can be adopted by the personskilled in the art to the particular pharmaceutically active substance,label or pharmaceutically active substance and label without anyadditional work. Examples of such reactions are described in Behrens CR, Liu B. Methods for site-specific drug conjugation to antibodies.MAbs. 2014 Jan-Feb; 6(1):46-53.

In theragnostic applications, i.e. in which the complex comprises both alabel and a pharmaceutically active substance, it is preferred that thelabel is covalently attached to the iron binding protein and thepharmaceutically active substance is non-covalently bound to the ironbinding protein and/or entrapped in the internal cavity formed uponassembly of the iron binding proteins.

In a third aspect the present invention relates to a method ofpreparation of the isolated targeted delivery system of the first orsecond aspect comprising the steps of

-   -   a) providing purified iron binding protein;    -   b) covalently or non-covalently linking a pharmaceutically        active substance, label or pharmaceutically active substance and        label to and/or encapsulating a pharmaceutically active        substance, label or pharmaceutically active substance and label        in an iron binding protein;    -   c) providing a CD45⁺ leukocyte cell; and    -   d1) incubating the CD45⁺ leukocyte cell in the presence of the        iron binding protein produced in step b) until the CD45⁺        leukocyte cell is at least partially loaded with the complex of        the iron binding protein and the a pharmaceutically active        substance, label or pharmaceutically active substance and label        produced in step b); and/or    -   d2) incubating CD45⁺ leukocyte cell in the presence of the label        until the CD45⁺ leukocyte cell is at least partially labelled        with the label.

In a further aspect the present invention relates to the isolatedtargeted delivery system producible by the method according to the thirdaspect of the present invention.

The formation of the adduct between the protein and the pharmaceuticallyactive substance and/or label is preferably by a non-covalent binding tothe iron binding protein and can be described as follows: In the case offerritin, pharmaceutically active substances and/or labels can betypically encapsulated within the internal cavity (physical confinement)by exploiting the association dissociation properties of the ferritinmacromolecule itself. pharmaceutically active substance and/or labelmolecules are held in place by non-covalent interactions with amino acidresidues within the cavity internal surface. Haemoglobin macromoleculesalso offer the possibility of non-covalent binding of selectedpharmaceutically active substances and/or labels molecules that may behosted within the heme binding pocket of haemoglobin itself. The heme inthe pocket can be displaced and be replaced by pharmaceutically activesubstances and/or labels with appropriate hydrophobicity profile. In afurther aspect, all proteins considered in the present invention may becovalently attached to pharmaceutically active substance and/or labelmolecules modified by specific active linker moieties reactive towardsthiol or amino groups of the protein itself. As such, ferritins orhaemoglobin may be linked to cysteine thiol reactive pharmaceuticallyactive substances and/or labels bearing a peptide based cleavable linker(e.g. cathepsin sensitive valine-citrulline sequence andpara-aminobenzylcarbamate spacer). As a notable example, the antimitoticagent monomethyl auristatin E (MMAE) has been used. The peptide-basedlinker binds the protein to the cytotoxic compound in a stable manner sothe drug is not easily released from the protein under physiologicconditions and help prevent toxicity to healthy cells and ensure dosageefficiency. The protein pharmaceutically active substance and/or labeladduct thus generated is capable of attaching to the selected receptortypes, i.e. CD163 for haemoglobin and TfR for ferritin or transferrin,respectively. Once bound the protein pharmaceutically active substanceand/or label adduct is internalised by endocytosis and thus selectivelytaken up by targeted cells. The vesicle containing the drug is fusedwith lysosomes and lysosomal cysteine proteases, particularly cathepsinB start to break down valine-citrulline linker and MMAE is no longerbound to the iron binding protein and is released directly into thetumour environment.

Alternatively, DM1-SMCC is an efficient mertansine derivative bearing alinker that specifically binds to lysine residues generating a covalentcomplex with ferritin, haemoglobin or transferrin in a reaction that hasbeen successfully described for antibodies. In particular, haemoglobin,ferritin or transferrin can be reacted with DM1-SMCC thus providing acovalent protein-drug adduct that can be cleaved inside cells andreleases the active drug in a time-dependent manner. The suppression ofmicrotubule dynamics by DM1 induces mitotic arrest and cell death.

The term “full load” is used in the context of the present invention torefer to the maximum amount of iron binding protein, preferablyferritin, complexed with a pharmaceutically active substance, label orpharmaceutically active substance and label that can be taken up by theCD45+ leukocyte cell, preferably macrophage more preferably activatedmacrophage.

In a fourth aspect the present invention relates to an isolated targeteddelivery system of the first aspect or second aspect of the invention orproducible according to the method of the third aspect of the inventionfor use as a medicament or diagnostic.

In a fifth aspect the present invention relates to a pharmaceutical ordiagnostic composition comprising the isolated targeted delivery systemof the first or second aspect of the present invention or producibleaccording to the method of the third aspect of the invention and apharmaceutically acceptable carrier and/or suitable excipient(s). Sincethe isolated targeted delivery system comprises living cells, it ispreferred that carriers and excipients are chosen in such to keep thecells alive.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

The term “carrier”, as used herein, refers to a pharmacologicallyinactive substance such as but not limited to a diluent, excipient,surfactants, stabilizers, physiological buffer solutions or vehicleswith which the pharmaceutically active substance is administered. Suchpharmaceutical carriers can be liquid or solid. Liquid carrier includebut are not limited to sterile liquids, such as saline solutions inwater and oils, including but not limited to those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. A saline solution is a preferred carrier whenthe pharmaceutical composition is administered intravenously. Examplesof suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

Suitable pharmaceutical “excipients” include starch, glucose, lactose,sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like.

“Surfactants” include anionic, cationic, and non-ionic surfactants suchas but not limited to sodium deoxycholate, sodium dodecylsulfate, TritonX-100, and polysorbates such as polysorbate 20, polysorbate 40,polysorbate 60, polysorbate 65 and polysorbate 80.

“Stabilizers” include but are not limited to mannitol, sucrose,trehalose, albumin, as well as protease and/or nuclease antagonists.

“Physiological buffer solution” include but are not limited to sodiumchloride solution, demineralized water, as well as suitable organic orinorganic buffer solutions such as but not limited to phosphate buffer,citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPESbuffer ([4 (2 hydroxyethyl)piperazino]ethanesulphonic acid) or MOPSbuffer (3 morpholino-1 propanesulphonic acid). The choice of therespective buffer in general depends on the desired buffer molarity.Phosphate buffer are suitable, for example, for injection and infusionsolutions.

The term “adjuvant” refers to agents that augment, stimulate, activate,potentiate, or modulate the immune response to the pharmaceuticallyactive substance comprised in the composition at either the cellular orhumoral level, e.g. immunologic adjuvants stimulate the response of theimmune system to the actual antigen, but have no immunological effectthemselves. Examples of such adjuvants include but are not limited toinorganic adjuvants (e.g. inorganic metal salts such as aluminiumphosphate or aluminium hydroxide), organic adjuvants (e.g. saponins orsqualene), oil-based adjuvants (e.g. Freund's complete adjuvant andFreund's incomplete adjuvant), cytokines (e.g. IL-1β, IL-2, IL-7, IL-12,IL-18, GM-CFS, and INF-γ) particulate adjuvants (e.g. immuno-stimulatorycomplexes (ISCOMS), liposomes, or biodegradable microspheres),virosomes, bacterial adjuvants (e.g. monophosphoryl lipid A, or muramylpeptides), synthetic adjuvants (e.g. non-ionic block copolymers, muramylpeptide analogues, or synthetic lipid A), or synthetic polynucleotidesadjuvants (e.g polyarginine or polylysine).

A striking observation was the disease specific homing of the targeteddelivery system of the present invention. The CD45⁺ leukocyte cellsappear to have a tropism for hypoxic areas and areas of oxidativestress. Hypoxia is a hallmark of various diseases as is oxidativestress. Accordingly, in a sixth aspect the present invention relates toan isolated targeted delivery system of the first aspect or secondaspect of the present invention or producible according to the method ofthe third aspect of the invention for use in preventing, treating ordiagnosing a disease characterized by hypoxic areas within the diseasestissue and/or by areas of oxidative stress (FIG. 27); tumours,preferably solid tumours and/or its metastases, preferably breastcancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, ora tumour having hypoxic areas, an inflammatory disease, inflamed tissue,preferably inflamed joints or arthritic joints, inflamed lung, inflamedintestine or other inflamed tissue; lymph nodes, preferably inflamedlymph nodes, or other non-physiological lymph nodes that develop duringdisease preferably but not only during infection, cancer, or autoimmunedisease; or ischemic areas, in particular in skin wounds or after organinfarctus (heart) or ischemic retina or for prophylactic or therapeuticvaccination, in particular to prevent or treat an infectious disease orcancer. This aspect also includes also of antigens to physiological ornon-physiological lymph nodes in order to vaccinate an individual or toinduce immune memory.

The term “treatment” as used herein includes all types of preventiveand/or therapeutic interventions medically allowed for the purpose ofcure, temporary remission, prevention, etc. for different purposesincluding delaying or stopping the progress of a disease, making alesion regress or disappear, preventing onset, or inhibiting recurrence.

The targeted delivery system according to the present invention enablestumour delivery of the pharmaceutically active substances, labels orpharmaceutically active substances and labels, which normally would notbe able to reach the tumour (for example, due to solubility problems).It also enables the delivery of pharmaceutically active substances,labels or pharmaceutically active substances and labels to the hypoxictumours or to the hypoxic areas of the tumour. This system also providesfor delivery of pharmaceutically active substance, label orpharmaceutically active substance and label to any area within anorganism subjected to hypoxic conditions, for example during ischaemicincidents, or undergoing an inflammatory process.

As mentioned above the present invention provides also the method fortargeted delivery into the tumour mass. This method comprisespreparation of CD45⁺ leukocytes, preferably activated macrophages whichenables highly efficient iron-binding protein (ferritin, haemoglobinand/or transferring) uptake by the macrophages, wherein said ferritin,haemoglobin and/or transferrin carry a pharmaceutically activesubstance, label or pharmaceutically active substance and label (forexample a drug/prodrug), tumour targeting and iron-binding proteintransfer to the cancer cell, where the pharmaceutically activesubstance, label or pharmaceutically active substance and label isreleased.

The present invention exploits CD45⁺ leukocytes, preferably activatedmacrophages loaded with iron-binding proteins linked with a drug/prodrugas a delivery system to target the tumour. Unsatisfactory response ofthe tumours to chemotherapy or difficulties in their detection usingimaging methods are mainly related to an altered penetration of theanticancer drugs to the hypoxic areas due to poor vasculature. However,these avascular regions attract CD45⁺ leukocytes, preferably activatedmacrophages to migrate even in areas far away from blood vessels.Therefore, they constitute a delivery system of particles to the tumourmass. A promising example of such particles is iron-binding protein.However, when used as single agents they do not reach hypoxic regions,similarly to other compounds and accumulate in other organs.

The present inventors linked anticancer drugs, hypoxia activatedprodrugs (for treatment purposes), labels or isotopes to haemoglobin ortransferrin using chemical methods and loaded it into CD45⁺ leukocytes(monocytes, macrophages, lymphocytes and/or granulocytes), preferablyactivated macrophages treating cells with iron-binding protein solutionas it is described in examples. The inventors observed that uponadministration to the animal, loaded CD45⁺ leukocytes, preferablyactivated macrophages migrate to the tumour hypoxic sites and releaseiron-binding protein with encapsulated pharmaceutically activesubstances, labels or pharmaceutically active substances and labels intothe cancer cells. This method allows precise administration of thepharmaceutically active substances, labels or pharmaceutically activesubstances and labels to the tumour site (especially to the hypoxicregions), avoiding their accumulation in other organs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Panel (A) shows a minimal active fragment of a consensus aminoacid sequence among mammalian ferritin H chains and two full lengthconsensus sequences based on several mammalian ferritin H chains (seeSEQ ID NO: 1, 2 and 7, respectively) as well as a minimal and fulllength amino acid sequence of mouse (SEQ ID NO: 3 and 4) and human (SEQID NO: 5 and 6) ferritin H chain. Panel (B) shows a minimal activefragment of a consensus amino acid sequence among mammalian ferritin Lchains and two full length consensus sequences based on severalmammalian ferritin L chains (see SEQ ID NO: 8, 9 and 14, respectively)as well as a minimal and full length amino acid sequence of mouse (SEQID NO: 10 and 11) and human (SEQ ID NO: 12 and 13) ferritin L chain.Panel (C) shows a minimal active fragment of a consensus amino acidsequence among mammalian haemoglobin alpha chains and one full lengthconsensus sequences based on several mammalian haemoglobin alpha chain(see SEQ ID NO: 15 and 16, respectively) as well as a minimal and fulllength amino acid sequence of human (SEQ ID NO: 17 and 18) haemoglobinalpha chain. Panel (D) shows a minimal active fragment of a consensusamino acid sequence among mammalian haemoglobin beta chains and a fulllength consensus sequences based on several mammalian haemoglobin betachain (see SEQ ID NO: 19 and 20, respectively) as well as a minimal andfull length amino acid sequence of human (SEQ ID NO: 21 and 22)haemoglobin beta chain. Panel (E) shows a N- and C-terminal minimalactive fragment of a consensus amino acid sequence among mammaliantransferrins (SEQ ID NO: 23 and 24) and a full length consensussequences based on several mammalian transferrins (SEQ ID NO: 25) aswell as a N- and C-terminal minimal active fragment of a humantransferrin (SEQ ID NO: 26 and 27) and full length amino acid sequenceof human transferrin (SEQ ID NO: 28). In the consensus sequences Xindicates a position that is variable and stands for any natural aminoacid. Preferably, in each case X in dependently of other X stands forthe amino acid present in the human protein.

FIG. 2: Shows macrophage inside the mouse tumour mass (TRITC stainedbefore injection, loaded with FITC-decorated ferritin).

FIG. 3: Shows confocal microscopy image of tumour tissue mouse injectedwith mammary cancer cells and given i.v. macrophages loaded withFITC-ferritin (asterix)—it is clearly observed, not only in macrophagesbut also in cancer cells, that ferritin-FITC spread within all tumourmass.

FIG. 4: Shows snapshots of one channel (in original green channelconverted to the grey-scale picture) recording using confocal microscopyof macrophages (indicated with *; loaded with FITC-ferritin) and cancercell (indicated with arrow; stained with red label and therefore notobserved in green channel before ferritin uptake) in vitro taken at thestarting time point (A) and after time long enough to fill cancer cellwith ferritin (B). FITC-ferritin was dynamically transported to thecancer cell (accumulating firstly in the vesicles; then spreading to thewhole cytoplasm as seen at this image (the cell appeared in greenchannel).

FIG. 5: Shows survival of mice receiving placebo and macrophages loadedwith ferritin-coupled melphalan and ferritin-coupled chlorambucil.

FIG. 6: Shows tumour cells apoptosis caused by treatment withcyclophosphamide and cyclophosphamide encapsulated in ferritins loadedto macrophages (given at the same doses).

FIG. 7: Shows MRI images of mouse mammary tumour. The mouse was treated(at time point 0 h) with macrophages (i.v. injection) loaded withferritin Fh. Then we observed increased diameter of blood vessels(arrow) filled with injected macrophages (giving significant T2-signalreduction) and afterword macrophages spread to the tissue (spot-likepattern; arrows). These changes (in the same time points) were observedin all examined mice.

FIG. 8: Shows ferritin, haemoglobin and transferrin uptake bymacrophages, ferritin and haemoglobin uptake by monocytes and ferritinuptake by lymphocytes and granulocytes.

FIG. 9: Shows the stability of the ferritin storage by macrophages.

FIG. 10: Shows transfer of ferritin, haemoglobin and transferrin frommacrophage to various cancer cells.

FIG. 11: Shows the transfer of ferritin from macrophage to cancer andnon-cancer cells.

FIG. 12: Shows the transfer of ferritin encapsulated with hypoxiaactivated prodrug from macrophage to cancer cells.

FIG. 13: Shows the apoptosis in cancer cells that received ferritin withencapsulated various anticancer agents from co-cultured macrophages orsoluble ferritin with the same agents.

FIG. 14: Shows the transfer of ferritin, haemoglobin and transferrinfrom monocyte to various cancer cells.

FIG. 15: Shows the transfer of ferritin and haemoglobin from granulocyteto various cancer cells.

FIG. 16: Shows the transfer of ferritin and haemoglobin from lymphocyteto various cancer cells.

FIG. 17: Shows the picture from two-photon microscopy showing tumourfrom a mouse that received pre-labeled (before administration)macrophages containing Ferritin-FITC.

FIG. 18: Shows the whole body imaging of mice that intravenouslyreceived labeled macrophages, showing their accumulation in the tumoursite and their distribution in other organs.

FIG. 19: Shows the migration of macrophages to hypoxic tissue, across-section of the tumour from a mouse that was administeredintravenously with pre-labeled macrophages, tumour hypoxic areas arevisualized with a hypoxia marker—pimonidazolone.

FIG. 20: Shows the presents localization of vesicles containingFITC-laded ferritin (round objects) in the microenvironment inside thetumour mass. Macrophages containing FITC-laded ferritin wereadministered intravenously to the mouse.

FIG. 21: Shows the signal recorded by PET from a whole-body analysis ofmice with the metastatic 4T1 cancer. Mice received intravenouslymacrophages loaded with ¹⁸F-FDG. Signal accumulation is increased in thelungs of mice with micrometastases (confirmed by pathology examination).Mice receiving plain ¹⁸F-FDG, or mice without 4T1 cancer had lower PETsignal. MQ-FDG: indicates mice with metastatic 4T1 tumour+intravenousmacrophages loaded with ¹⁸F-FDG. FDG: indicates mice with metastatic 4T1tumour+intravenous free ¹⁸F-FDG. Naive mice MQ-FDG: Indicates naive micewithout tumour+intravenous macrophages loaded with ¹⁸F-FDG.

FIG. 22: Shows the tumour homing by different CD45+ leukocyte cell typesusable in the targeted delivers systems of the invention. Mice with CT26tumors (marked by the arrows) were intravenously injected with PBS orfluorescently labelled cells. Panel A. —PBS, no cells added, PanelB.—shows homing of a CD45⁺ leukocyte subpopulation, Panel C. showshoming of activated CD4⁺ T lymphocytes, Panel D. shows homing of amacrophage-monocytic cell line. 24 hours after injection mice wereanaesthetized and imaged. Panel E. Shows the quantification of thetumour homing by different CD45+ leukocyte cells.

FIG. 23: Shows Survival of mice with CT26 tumor receiving therapy withmonocytes loaded with ferritin-Cisplatin. n=6-8 mice/group. Survival ofthe mice was calculated by reaching (1500 mm3) maximum-allowed tumourvolume (measured by caliper, according to formula A×B2).

FIG. 24: Shows targeting of intravenously administered cells to inflamed(arthritic) joints Control mouse (panel A.) or a mouse with severeinflammation of the joints (panel B.) have been intravenously injectedwith macrophage-monocyte cell line. Before injection cells were labelledwith fluorescent tracer. 24 hours after injection joints were removedand single cell suspension generated. Cells have been analyzed by flowcytometry. Fluorescently labelled cells are presented in the P2 area.Panel C. shows quantification of the fluorescent cells in the inflamedthan in control joints. Macrophage-monocyte cells are present withgreater number in the inflamed than in control joints.

FIG. 25: Shows targeting of intravenously administered cells to thelymph nodes. Control mouse (panel A.) or a mouse with severeinflammation of the joints (panel B.) have been intravenously injectedwith activated macrophages (with M-CSF and CCL2). Before injection cellswere labelled with fluorescent tracer. 24 hours after injection micewere anaesthetized and imaged. Mice with inflammatory condition havepositive signal derived from cell-tracer, from the lymph nodes (markedby white arrows).

FIG. 26: Shows radiolabeling of monocyte-macrophage cell line with plain⁸⁹Zr-oxinate, i.e. without iron binding protein (˜1.85 MBq) for PETimaging. Radioactivity of cells after loading with ⁸⁹Zr is indicated inMBq.

FIG. 27: Ferritin transfer was examined in cancer cells (EMT6)co-cultured with ferritin-loaded macrophages (RAW264.7) for 2 hrs. Threeexperimental conditions were used: “ctrl” (normal co-culture, nostress); “EMT6 OS” when EMT6 cells were subjected to the oxidativestress before co-culture and then co-cultured with macrophages atnormoxic conditions and “co-culture OS” when both cell types weresubjected to the oxidative stress before the co-culture. Results showedthat cancer cells subjected to the oxidative stress received (frommacrophages) more ferritins than control cells. The uptake was even moreefficient when both cell types were subjected to the oxidative stress.It can be related with higher load of ferritin due to oxidative stressand therefore more efficient transfer, or oxidative stress triggersmacrophages to more efficiently deliver ferritins to cancer cells inorder to reduce oxidative stress in neighboring cells. Panel A shows:Ferritin uptake from macrophages (2 hrs co-culture) in oxidative stress(oxidative glucose treatment for 16 hrs). Panel B shows: Ferritin uptakefrom macrophages (2 hrs co-culture) in oxidative stress (oxidativeglucose treatment for 16 hrs).

EXAMPLE SECTION Example 1—Activation of Macrophages

Macrophages for use according to the present invention were obtained,differentiated and activated as follows. In order to activatemacrophages, they are obtained firstly from bone marrow precursors (forexample see paper: Weischenfeld and Porse, 2008, CSH Protoc, doi.10.1101/pdb.prot.5080) or blood monocytes. Alternatively, they can beobtained from peritoneum. The methods of macrophage isolation, culture,differentiation and polarization/activation are well known for thoseskilled in the art. For example, they have been described in details byMurray et al. (Immunity, 2014, 41(1):14-20).

In this practical realization of the invention bone marrow derivedmacrophages were obtained from BALB/c or C57Bl/6 mouse, however canineblood-monocyte-derived macrophages or commercially available macrophagecell lines (monocyte-macrophage lineage mouse cells: RAW 264.7, J744,human: THP-1, U937, or canine DH82 cell line).

Shortly, such bone marrow derived macrophages are seeded in plasticPetri dish in 5 ml medium (3 ml cells per plate):DMEM:F12+glutamine/glutamax+10% FBS+Penicillin/Streptomycin and 20% ofL929 conditioned medium or M-CSF (50 ng/ml). In the next five days themedium is supplemented in growth factor and one of the activatingcompounds or their combinations as one cytokine cocktail.

Alternatively, macrophages have been cultured in “M1/M2 MacrophageGeneration Medium” (Promocell) or equivalent commercially available orself-made medium containing all the necessary cytokines and interleukinsto consider them as activated.

In order to obtain macrophages from blood monocytes, fresh blood (notolder than 12 hours) cells were purified by centrifugation usingHistopaque system 1.077 g/ml or equivalent and white blood cells (oralternatively, only white blood cells collected from the blood bank) inan appropriate amount of pre-warmed Monocyte Attachment Medium (orequivalent, e.g. DMEM/RPMI supplemented with M-CSF), e.g. 15 ml Mediumper T-75 flask. A seeding density should be of 1-2 million/cm² formononuclear cells with a monocyte content of ≥2 5% and 1.5-3 million/cm²for a monocyte content of <25%. Then, cells are incubated for 1-1.5hours at 5% CO₂ and 37° C. in the incubator without any furthermanipulation.

After cell attachment, they are washed at least twice, and then anappropriate amount of complete “M1- or M2-Macrophage Generation MediumDXF” is added to the cells (e.g. 20 ml per T-75 flask) and cells areincubated for 6 days at 37° C. and 5% CO₂ without medium change. Inorder to activate macrophages, the whole medium is replaced with mediumsupplemented with activating compound.

Activating compounds used in this invention (for bone-marrow derivedcells or to activate cells from monocyte-macrophage cell lines) are asfollows: IL-4 (20 ng/ml), IFN-γ (at least 20 ng/ml), LPS (at least 10ng/ml), IL-13 (at least 20 ng/ml), IL-10 (at least 20 ng/ml),dexamethason (at least 20 μg/ml), CCL2 (at least 20 ng/ml), oxLDL (atleast 20 ng/ml), TNF-α (20 ng/ml), TGF-β (20 ng/ml), cortisol (150-300ng/ml) or their combinations as one cytokine cocktail. In order toobtain non-activated macrophages, the activating compound has not beenadded.

Reverse of the polarization/activation of macrophages (from classicallyactivated to alternatively activated) can be reached for example byculture of macrophages in appropriate cytokines listed above for atleast 48 hours.

Example 2—Monocyte Isolation

In order to obtain monocytes in this practical realization of theinvention bone marrow derived or spleen-derived monocytes were obtainedfrom BALB/c or C57Bl/6 mouse, however canine blood monocyte orcommercially available monocyte cell lines were used(monocyte-macrophage lineage mouse cells: RAW 264.7, J744, human cells:THP-1, U937, or canine cells DH82).

To obtain blood monocytes, fresh blood (not older than 12 hours) cellswere purified by centrifugation using Histopaque system 1.077 g/ml orequivalent and white blood cells are seeded in an appropriate amount ofpre-warmed Monocyte Attachment Medium (or equivalent, e.g. DMEM/RPMIsupplemented with 20 ng/ml M-CSF), e.g. 15 ml Medium per T-75 flask.Alternatively, only white blood cells collected from the blood bank(buffy coat) may be used. After cell attachment, they are washed atleast twice, and adherent cells are considered as monocytes.

In order to obtain bone-marrow derived monocytes, in this practicalrealization of the invention monocytes were obtained from BALB/c orC57Bl/6 mouse. Shortly, such bone marrow derived precursors wereisolated using Mouse Monocyte Enrichment Kit (StemCells) according tomanufacturer's instructions. In order to obtain spleen derivedmonocytes, in this practical realization of the invention, the spleenhas been mechanically dissociated to obtain single cell suspension andpassed through the 70 μm cell strainer. Cells were centrifuged andsupernatant was removed. Optionally erythrocyte lysis was performedprior to monocyte isolation by EasySep™ Mouse Monocyte Enrichment Kit(StemCells) according to manufacturer's instructions.

To obtain better effects of their protein loads and migration before usethey may be pre-treated with activation stimuli: IL-4 (20 ng/ml), IFN-γ(at least 20 ng/ml), LPS (at least 10 ng/ml), IL-13 (at least 20 ng/ml),IL-10 (at least 20 ng/ml), dexamethason (at least 20 μg/ml), CCL-2 (atleast 20 ng/ml), oxLDL (at least 20 ng/ml), TNF-α (20 ng/ml), TGF-β (20ng/ml), cortisol (150-300 ng/ml) or their combinations as one cytokinecocktail.

Example 3—Granulocyte Isolation

To obtain granulocyte cells from blood, 9 parts of blood were dilutedwith 1 part of ACD buffer (containing 0.17 M d-glucose, 0.10 M citricacid, 0.11 M trisodium citrate). Blood from this step was furtherdiluted with PBS at the 1:1 ratio and centrifuged. After removing plasmaand buffy coat, remaining cells were mixed with PBS to 80% of theoriginal volume from the first step (ACD-blood) and then diluted withcold distillated water at the ratio of 4:12. Then, 6 parts of 2.7% ofNaCl solution were added and centrifuged. After removal of supernatantcells were resuspended in RPMI-1640 medium. These cell were consideredas granulocytes.

Example 4—Lymphocyte Isolation

In order to obtain spleen derived lymphocytes, in this practicalrealization of the invention, the spleen has been mechanicallydissociated to obtain single cell suspension and passed through the 70μm cell strainer. Cells were centrifuged and supernatant was removed.After erythrocyte lysis, lymphocytes were isolated using a negative cellisolation strategy with a help of EasySep™ CD4⁺ Enrichment Kit(Stemcell) according to manufacturer s instructions.

Lymphocytes were activated by the 3-5 day culture in RPMI mediumsupplemented with glutamine/glutamax, 10% FBS, Penicillin/Streptomycin,in the presence of beads covered with anti-CD3 and anti-CD28 (Gibco)according to manufacturer's instructions.

Activation of lymphocytes was confirmed with upregulation of CD25 andCD69 cell surface expression, that was monitored by flow cytometry.

Example 5—Leukocyte (CD45⁺ Cells) Isolation

In order to obtain macrophages from blood monocytes, fresh blood (notolder than 12 hours) is purified by centrifugation using Histopaquesystem 1.077 g/ml or equivalent and white blood cells (or alternatively,only white blood cells collected from the blood bank) are used forfurther steps of the procedure. Alternatively, leukocytes are obtainedafter erythrocyte lysis of the whole blood, as it was done in thispractical realization of the invention.

Example 6—Preparation of Ferritin Complexes

In order to incorporate ferritins with the anticancer drug (e.g. classicdrugs like cyclophosphamide, chlorambucil, melphalan, bendamustine,banoxantrone or hypoxia-activated prodrug like TH-302) or with the“imaging contrast agents” (e.g. ferrihydride or isotope) ferritins haveto be prepared before macrophage treatment. Shortly, recombinant mouseproteins according to SEQ ID NO: 4 (FIG. 1) are obtained as follows. Theexpression vector pET-22b containing a synthetic gene encoding ferritinprotein of SEQ ID NO: 4 was transformed into E. coli BL21 (DE3). E. coliculture was grown at 37° C. to OD600 0.6 in 1 L of Luria-Bertani broth(LB) added with ampicillin (100 mg/L). Protein expression was induced byaddition of 1 mM isopropyl thio-b-D-galactoside (IPTG) and the culturewas incubated overnight. Cells were harvested by centrifugation (15000 gfor 15 min) and suspended in 20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1mg/mL DNase, 10 mM MgCl₂ and disrupted by sonication. The lysate wascentrifuged at 15000 g for 30 min and the supernatant was treated 10 minat 50° C., centrifuged to remove denatured proteins and then at 70° C.for 10 min and centrifuged again. The supernatant was added with 30%(NH)₄SO₄ at 4° C. stirring for 1 h and centrifuged at 15000 g for 30min. The supernatant was added with 70% (NH)₄SO₄ at 4° C. stirring for 1h and centrifuged at 15000 g for 30 min. The pellet was resuspended in20 mM Hepes (pH 7.5), 150 mM NaCl and dialysed overnight at 4° C.against the same buffer. The protein was loaded on a HILOAD 26/600SUPERDEX 200 gel-filtration column (GE-Healthcare) and then sterilefiltered and stored at 4° C. (FIG. 9) Protein concentration wasdetermined spectrophotometrically at 280 nm using a molar extinctioncoefficient of 21000 M⁻¹ m⁻¹ and by Bradford assay measuring theabsorbance at 595 nm.

Said ferritins include recombinant mammalian ferritin proteins H and/orL homopolymers.

Ferritins, obtained as previously described, are purified by standardmethods in order to obtain an endotoxin free, pre-clinical grade product(see, for example: Ceci et al. 2011, Extremophiles 15(3):431-439;Vanucci et al. 2012, Int J Nanomed 7:1489-1509). Shortly, the ferritinconserved sterile in a storage solution containing 20 mM Hepes pH 7.5 isdiluted to a final concentration of 4 uM in 24-mer in acidic solution(final pH<3.0) or, alternatively, at highly basic pH values (pH>9.5)(see for example Pontillo et al., 2016), thus allowing the dissociationof multimer. Drugs are dissolved at very high concentrations in theappropriate solvent and then a small volume is added to the ferritinsolution with a 200 molar excess. PH is then brought to neutrality byaddition of appropriate amounts of NaOH/HCl solutions in order to allowmultimer reconstitution. Current experimental methods indicate thatthree/four washings using PBS (concentration steps) in 100 kDa cut offconcentrators allows rapid and complete elimination both the co-solventsas well as nonencapsulated drugs and full recovery of drug loadedferritin nanocages. The ferritin-drug complex thus obtained was thenflash freezed in liquid nitrogen and lyophilised.

Depending on the choice of co-solvent and on the intrinsic chemicalproperties of the drug molecule, it can be estimated that up to 150-180drug molecules can be entrapped/adsorbed within the 24-mer ferritincage.

Pharmaceutically active substances or labels may also be covalentlycoupled to ferritin amino acid side chains (lysines or cysteines) byappropriate choice of phenylhydrazone, succinimide or maleimideactivated drugs. Accordingly, i) phenylhydrazone derivative may breaksand liberates the drug from the ferritin surface, ii) lysine boundderivatives may become active after full protein degradation intoaminoacids or iii) cysteine bound derivative may be liberated within thecell through reductive hydrolysis of the maleimede thioether link.

Example 7—Preparation of Haemoglobin-Compound Complex

Human haemoglobin is prepared from fresh red cells as described inRossi-Fanelli et al. (Archives of biochemistry and biophysics77:478-492, 1958). Shortly, the heparinized blood, obtained from healthydonors, was centrifuged at 1600 rpm for 30 minutes (4° C.) to sedimentthe RBCs. Buffy coat was accurately removed by needle aspiration on thesurface of the pellet. The plasma supernatant was discarded and the RBCpellet was washed three times by resuspending the RBCs in isotonic 0.9%saline solution and centrifuging at 1600 rpm for 30 minutes at 4° C.After the final saline wash and centrifugation step, the RBC pellet wasresuspended in distilled water buffered at pH 7.2 with 5 mM potassiumphosphate buffer (PB, pH=7.2) and allowed to lyse at 4° C. overnightunder gentle stirring. Dialyzed RBC lysate was subsequently centrifugedat 13.000 rpm for 30 min at 4° C. and supernatant was directly loaded onan ÄKTA Explorer system equipped with an XK 26/40 column packed withQ-sepharose XL resin (GE Healthcare) at room temperature. Columns wereequilibrated with buffer A (20 mM Tris-HCl, pH=8.2) at a flow rate of 12mL/min and washed three times with the same buffer. A linear gradientelution was generated by changing from 100% buffer A to 75% buffer B (20mM Tris-Cl, plus 0.2 M NaCl pH8.20) followed by a step gradient of 100%buffer B. Upon elution, a fraction collector was used to collect proteinfractions. Protein thus obtained was analyzed by SDS page and storedfrozen at −80° C.

Human Haemoglobin (SEQ ID NO: 18 or 22, see FIG. 1) can be readilycovalently linked to appropriate drug conjugates, host hydrophobic drugmolecules within the heme binding pocket or even transport smallcytotoxic molecules linked to the heme iron. Hb can be easily modifiedby selective attachment of the appropriate drug conjugate to thecysteine residue in position 93 of the beta chains, the only titratablecystein on the protein surface. Maleimido functionalized drugs, such asthe tubuline inhibitor MonomethylAuristatin (MMAE) or the succinimidefunctionalized mertansine analogue (DM1-SMCC) are most notable examplesof extremely potent cytotoxics that can be readily and specificallyattached to the relevant cys beta93 residue (for maleimidofunctionalized drugs) or to one or more lysine residues (succinimidefunctionalized drug), respectively. These drugs have been convenientlyconjugated to human haemoglobin according to the following procedures:

The auristatin E analogue,maleimidocaproyl-valine-citrulline-paminobenzoyloxycarbonyl-monomethylauristatin E (vcMMAE) was obtained from MedChem Express (Princeton,N.J.). The Haemoglobin vcMMAE adduct was prepared as follows. Humanhaemoglobin solution was adjusted to a concentration of 120 μM heme withreaction buffer (50 mM phosphate buffer pH 6,8, containing 0.1 mM EDTA)and conjugated with 10-fold molar excess of vcMMAE in the presence of20% v/v acetonitrile solution at 4° C. overnight. Maleimide groups reactefficiently and specifically with free (reduced) sulfhydryls at pH6.5-7.5 to form stable thioether bonds. The excess vcMMAE was purifiedand buffer-exchanged with D-PBS using PM 100 ultrafiltrationconcentrator. The yield of conjugation was approximately 80% of thetotal cysteines. Formation of vcMMAE conjugate was confirmed by LC-MSanalysis and by titration of residual free thiol group withp-chloromercuribenzoate. The concentrations of Hb-vcMMAE conjugates weredetermined by UV-vis spectroscopy analysis.

The mertansin analogue DM1 SMCC (Alb Technology Ltd, Hederson, Nev.,USA), functionalized for lysine covalent attachment, was prepared asfollows. Human haemoglobin solution was adjusted to a concentration of400 μM heme with reaction buffer (0.1 mM phosphate buffer pH 7.4,containing 0.5 mM EDTA) and conjugated with 20-fold molar excess ofDM1-SMCC in the presence of 10% v/v DMSO solution at 4° C. for 16 hours.The amine-reactive succinimidyl ester couples to amines thus yielding acovalent adduct with lysine groups on the surface of the protein. Theexcess DM1-SMCC was eliminated and buffer-exchanged with D-PBS using PM100 ultrafiltration concentrator. The yield of conjugation wasapproximately 2.4 mertansine molecules per haemoglobin tetramer.Formation of DM1-SMCC conjugate was confirmed by LC-MS analysis. Theconcentrations of Hb-DM1-SMCC conjugates were determined by UV-visspectroscopy analysis.

Alternatively, the apo-protein solution must be kept on an ice bath forthe duration of the reconstitution process. A 1.5-fold molar excess ofCuT-CPP (Cu-TCPP4,4′,4″,4′″-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)—Cu⁶⁷) in0.1 M NaOH must be added dropwise to the apo-globin solution in 0.2 MKPi buffer at pH 7.0 vortexed quickly at room temperature and thenplaced back in ice bath for 30 minutes. The protein solution must thenbe filtered in a syringe filter before use.

Example 8—Preparation of Transferrin-Compound Complex

The serum was obtained from healthy donor and excess iron was added inthe presence of citrate ions as a chelator and bicarbonate, which isfacilitates for iron binding to transferrin. The reaction mixturecontained 6.5 mg sodium bicarbonate and 153.16 ferric citrate in pH=8,4° C., 1 hour per 100 mL of serum. Albumin was subsequently precipitatedby Rivanol (4%) by adding the alcohol solution to the serum sample in a3.5 V/V ratio at 4° C., and pH=9.4 for 2 h. Then, the solution wascentrifuged at 3000 rpm for 20 min and finally filtered by filter on a0.8 mm syringe filter. Excess Rivanol was subsequently removed bygel-filtration on a Sephadex G-25 column in ammonium sulfate 0.025 M. Afirst precipitation of by saturated ammonium sulfate 50% at pH=6.5 wassubsequently carried out followed by centrifugation at 3000 rpm for 10min (immunoglobulin removal). A second precipitation at 80% saturatedammonium sulfate was then carried out thus allowing recovery oftransferrin the precipitate. Solid precipitate was then dissolved inbuffer of 0.06 M Tris HCl buffer, pH=8, containing 1 M NaCl. Thesolution was dialyzed in the same buffer to allow full removal ofammonium sulfate. Protein solution was then concentrated with acentricon PM50 centrifugal concentrator up to 10-15 mg/ml (as estimatedby Bradford method) and loaded on a Sephadex G-100 gel-filtration column(2,4×80 cm) equilibrated in 1M NaCl, flow rate of 15 ml/h. Transferrinthus obtained was estimated to be 88-90% pure by SDS page. Ion-exchangechromatography by anion exchanger DEAE Sephadex A-50 was then used as afinal polishing step. The transferrin sample was loaded in the columnequilibrated with 0.06 M Tris HCl at pH=8 and eluted by a linearconcentration gradient with elution buffer, 0.3 M Tris HCl, pH=8.Protein purity was higher than 98% with a yield of about 150 mg per 100mL of serum.

Human Holo-transferrin, (SEQ ID NO: 28, FIG. 1) similarly to haemoglobincan be readily covalently linked to appropriate drug conjugates,although there is only availability for lysine modifications, due to theabsence of freely titratable cysteine groups. Thus the succinimidefunctionalized mertansine analogue (DM1-SMCC) has been used tocovalently attach to one or more lysine residues (succinimidefunctionalized drug). The drug has been conveniently conjugated totransferrin according to the following procedure:

The mertansin analogue DM1 SMCC (Alb Technology Ltd, Hederson, Nev.,USA), functionalized for lysine covalent attachment, was prepared asfollows. Transferrin solution was adjusted to a concentration of 100 μMheme with reaction buffer (0.1 mM phosphate buffer pH 7.4, no EDTA inthis case due to possible iron chelation effects) and conjugated with20-fold molar excess of DM1-SMCC in the presence of 8% v/v DMSO solutionat 4° C. for 16 hours. The amine-reactive succinimidyl ester couples toamines thus yielding a covalent adduct with lysine groups on the surfaceof the protein. The excess DM1-SMCC was eliminated and buffer-exchangedwith D-PBS using PM 100 ultrafiltration concentrator. The yield ofconjugation was approximately 1.5 mertansine molecule per transferringdimer. Formation of DM1-SMCC conjugate was confirmed by LC-MS analysis.The concentrations of Transferrin-DM1-SMCC conjugates were determined byUV-vis spectroscopy analysis.

Alternatively, the apo-protein solution is kept in an ice bath for theduration of the reconstitution process. A 1.5-fold molar excess ofCuT-CPP (Cu-TCPP4,4′,4″,4′″-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)—Cu⁶⁷) in0.1 M NaOH must be added dropwise to the apo-globin solution in 0.2 MKPi buffer at pH 7.0 vortexed quickly at room temperature and thenplaced back in ice bath for 30 minutes. The protein solution must thenbe filtered in a syringe filter before use.

Example 9—Obtaining Ferritin Loaded Cells

Obtained cells are incubated in ferritin solution for a time and at theconcentration sufficient to ensure proper ratio of ferritin/cell fortheir full load and also to ensure proper drug content to obtaintherapeutic effect or contrast for proper imaging. The time andconcentration may vary depending on the number of moleculesencapsulated/adsorbed into the ferritin cage, status of cell activation,condition and number of their intended administration.

For example, to ensure proper load with ferritins, cells are incubatedfor 1-4 hours in ferritin solution 0.8 mg/ml in standard cultureconditions. The frame of ferritin concentration may vary at leastbetween 0.01 and 4 mg/ml as well as incubation time (5 min-6 hours ormore). Adjusting time and concentration of ferritin load to cells, theinfluence of ferritin and treatment conditions on cell viability shouldbe minded. Cells obtained as stated above very easily uptake ferritinsin a relatively short time (in minutes; FIG. 8). Once they absorbferritins, they do not release it to the culture medium (FIG. 9).

Nevertheless, the person skilled in the art is able to re-adjust theabove conditions and optimize the protocol for the own purposes in theown laboratory.

Example 10—Obtaining Haemoglobin Loaded Cells

Obtained cells are incubated in haemoglobin solution for a time and atthe concentration sufficient to ensure proper ratio of haemoglobin/cellfor their full load and also to ensure proper drug content to obtaintherapeutic effect or contrast for proper imaging. The time andconcentration may vary depending on the number of molecules linked withthe haemoglobin molecule, status of cell activation, condition andnumber of their intended administration.

For example, to ensure proper load with haemoglobins, cells areincubated for 1-4 hours in haemoglobin solution 0.1 mg/ml in standardculture conditions. The frame of haemoglobin concentration may vary atleast between 0.01 and 0.2 mg/ml as well as incubation time (5 min-4hours or more). Adjusting time and concentration of haemoglobin load tocells, the influence of ferritin and treatment conditions on cellviability should be minded. Cells obtained as stated above very easilyuptake haemoglobins in a relatively short time (in minutes; FIG. 8).

Nevertheless, the person skilled in the art is able to re-adjust theabove conditions and optimize the protocol for the own purposes in theown laboratory.

Example 11—Obtaining Transferrin Loaded Cells

Obtained cells are incubated in transferrin solution for a time and atthe concentration sufficient to ensure proper ratio of transferrin/cellfor their full load and also to ensure proper drug content to obtaintherapeutic effect or contrast for proper imaging. The time andconcentration may vary depending on the number of molecules linked withthe transferrin molecule, status of cell activation, condition andnumber of their intended administration.

For example, to ensure proper load with transferrins, cells areincubated for 1-4 hours in transferrin solution 0.1 mg/ml in standardculture conditions. The frame of transferrin concentration may vary atleast between 0.01 and 0.2 mg/ml as well as incubation time (5 min-4hours or more). Adjusting time and concentration of transferrin load tocells, the influence of transferrin and treatment conditions on cellviability should be minded. Cells obtained as stated above very easilyuptake transferrin in a relatively short time (in minutes; FIG. 8).

Nevertheless, the person skilled in the art is able to re-adjust theabove conditions and optimize the protocol for the own purposes in theown laboratory.

Example 11—Ferritin/Haemoglobin/Transferrin-Macrophage Complex as UsefulDelivery Tool to Cancer Cells

The macrophages from Example 1 prepared as described in Examples 8, 9and 10, very easily transport ferritins, haemoglobins, transferrins tothe cancer cells: mouse mammary cancer, colon cancer, canine mammarycancer, human breast, pancreatic, and bladder cancer (FIGS. 4, 10).Moreover, this transfer is much more specific to cancer cells than tonon-cancer cells (FIG. 11). However, in case of cancer cells the ratioof both cell types is crucial. The more macrophages the better andfaster the transport is. The most efficient transfer to the cancer cellswas observed when ratio of macrophages to cancer cells was 1:1 or more.

This transfer occurred not only when the protein carriers are conjugatedwith fluorescent label (e.g. FITC or Alexa610), but also when they wereconjugated/encapsulated with other compounds, e.g. anticancer drugs(FIG. 12 shows this transfer of ferritin encapsulated with fluorescenthypoxia activated prodrug—banoxantrone). This transfer of compoundsconjugated with anticancer drugs made the effect inducing apoptosis incancer cells (FIG. 6, 13).

Example 12—Ferritin/Haemoglobin/Transferrin-Monocyte Complex as UsefulDelivery Tool to Cancer Cells

The monocytes from Example 2 prepared as described in Examples 8, 9 and10, very easily transport ferritins, haemoglobins, transferrins to thecancer cells (FIG. 14). However, the ratio of both cell types isimportant. The more monocytes the better and faster the transport is.The most efficient transfer to the cancer cells was observed when ratioof monocytes to cancer cells was 1:1 or more.

Example 13—Ferritin/Haemoglobin/Transferrin-Granulocyte Complex asUseful Delivery Tool to Cancer Cells

The granulocytes from Example 3 prepared as described in Examples 8, 9and 10, very easily transport ferritins, haemoglobins, transferrins tothe cancer cells (FIG. 15). However, the ratio of both cell types iscrucial. The more granulocytes the better and faster the transport is.The most efficient transfer to the cancer cells was observed when ratioof granulocytes to cancer cells was 1:1 or more.

Example 14—Ferritin/Haemoglobin/Transferrin-Lymphocyte Complex as UsefulDelivery Tool to Cancer Cells

The lymphocytes from Example 4 prepared as described in Examples 8, 9and 10, very easily transport ferritins, haemoglobins, transferrins tothe cancer cells (FIG. 16). However, the ratio of both cell types iscrucial. The more lymphocytes the better and faster the transport is.The most efficient transfer to the cancer cells was observed when ratioof lymphocytes to cancer cells was 1:1 or more.

Example 15—Cell Labeling with Radioisotope

In this invention cells were labeled with ¹⁸F-FDG or ⁸⁹Zr-oxinate inorder to become imaged on PET. Cells have been detached from the plateand incubated with ¹⁸F-FDG or ⁸⁹Zr-oxinate solution in adequateconcentration to ensure the most optimal ¹⁸F-FDG or ⁸⁹Zr-oxinate uptakeby cells allowing their radio-detection at the site of theiraccumulation. In this practice of the invention cells were incubated atroom temperature for at least 90 min. in ⁸⁹Zr-oxinate solutioncontaining 3-9 MBq per 5 min of cells. However, the ratio ofradioisotope and cells influences significantly the reaction efficacy(FIG. 26). After labeling, cells are be centrifuged and supernatant isremoved. This step should be repeated until no radioactivity is detectedin the supernatant.

Example 16—Ferritin/Haemoglobin/Transferrin/Label-Leukocyte Complex asUseful Delivery Tool to Tumor, Arthritic Joints and Lymph Nodes

The macrophages from Example 1 prepared as described in Examples 9, 10,11 and 11; monocytes from Example 2 prepared as described in Examples 9,10, 11 and 12, granulocytes from Example 3 prepared as described inExamples 9, 10, 11 and 12, and lymphocytes from Example 4 prepared asdescribed in Examples 9, 10, 11 and 12, highly efficiently migrate tothe tumors (FIG. 22 and FIG. 23), arthritic joints (FIG. 24), in thisparticular example monocytic-macrophage cell line was used) and inflamedlymph nodes (FIG. 25), in this particular example monocyte/youngmacrophages activated using M-CSF and CCL-2 were used). Once theymigrate to the tumors or other tissues they very easily transportferritins, haemoglobins, transferrins and labels to the tumor in theenough amount to be detected using imaging systems (FIGS. 2, 3, 7, 17,and 18).

Example 17—Leukocyte-Protein Carrier Complex as Useful TargetedPharmaceutically Active Substance Delivery System to Hypoxic Regions

Macrophages prepared as above are injected into the tail vein of animalwith the tumour (appropriate number of macrophages should be adjusted tothe tumour size, stage of development and presence of metastases). As itis shown on FIGS. 2, and 17 they specifically reach the tumour (after afew hrs) and also disperse in other organs of the whole animal (FIG.18). Moreover, as it is shown on FIG. 19, in hypoxic model they are alsoable to migrate to the avascular and hypoxic sites and to transfercarrier proteins to cancer cells (FIGS. 3, and 20).

For the imaging purposes, 1-50 millions of macrophages were injectedinto the tail vein of mammary or colon cancer tumour-bearing animal.Before, macrophages were pre-labeled with Cell Tracker and loaded withferritin-FITC (as shown in Example 8). Using two-photon of the tumourmass 8 hours after administration of macrophages the presence ofmacrophages carrying Ferritin-FITC was detected (FIG. 17). Theirspecific targeting of tumour but also their migration to other organswas shown using whole animal body imaging (IVIS) after macrophagepre-labeling using DIR cytoplasmic dye (FIG. 18).

The 1-10 millions of macrophages loaded with ferritin encapsulatedcyclophosphamide, melphalan and ferritin encapsulated chlorambucil wereinjected i.v. into the tumour-bearing mice (300 000-500 000 of EMT6cells injected into the skin flank). We made 3 injections of macrophagesevery third day (on the day 5, 8 and 11 after cancer cells injection oron the day 7, 10 and 13 after cancer cells injection) or fiveconsecutive injections every day and we observed increased mousesurvival (FIG. 5).

Example 18—Leukocyte-Protein Carrier Complex or Labeled Leukocyte asUseful Targeted Label Deliver Agent

The targeting of the targeted delivery system described in presentinvention can be followed by coupling the ferritin to a contrast agent.As it is presented on FIG. 21, after injection of 1-50 ml of macrophagesloaded with ferritin coupled as described in Example 8 with a contrastagent (in this case: ferrihydrite, however the same results are obtainedwith isotope, e.g. ¹²³I) or labeled with isotope (in this case⁸⁹Zr-oxide or ¹⁸F-FDG) (FIG. 21) they can be easily detected by MRI, PETor SPECT. In this example (FIG. 7), mammary-tumour bearing mice wereimaged using MRI at 3, 22 and 24 hours after i.v. injection ofmacrophages loaded with ferritin Fh. The mouse was treated (at timepoint 0 h) with macrophages. Then increased diameter of blood vessels(arrow) filled with injected macrophages (giving significant T2-signalreduction) has been observed and afterword macrophages spread to thetissue (spot-like pattern; arrows). These changes (in the same timepoints) were observed in all examined mice.

Macrophages were also labeled with ⁸⁹Zr-oxide or ¹⁸F-FDG (5-50 min) andtheir radioactivity was confirmed to be imaged using PET after i.v.administration to the tumour-bearing mice. These mice were inoculatedwith 4T1 metastatic cell line 3 weeks before the experiment andmetastases in the lungs, liver and spleen were histopatologicallyconfirmed. At FIG. 21 it is seen that macrophages migrated to theregions with metastatic tumours allowing their visualization at PET.

While the foregoing written description of the invention enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific embodiment, method, and examples herein. The inventionshould therefore not be limited by the above described embodiment,method, and examples, but by all embodiments and methods within thescope and spirit of the invention.

1. An isolated targeted delivery system comprising a CD45⁺ monocyte,CD45⁺ monocyte-macrophage, CD45⁺ lymphocyte and/or CD45⁺ granulocyte(commonly referred to as “CD45⁺ leukocyte cell”) comprising within saidcell a complex of one or more iron binding proteins and apharmaceutically active substance, label or pharmaceutically activesubstance and label.
 2. An isolated targeted delivery system comprisinga CD45⁺ leukocyte cell comprising one or more label.
 3. The isolatedtargeted delivery system according to claim 1, wherein the CD45⁺leukocyte cell is producible from a CD34⁺ hematopoietic precursor cell.4. The isolated targeted delivery system according to claim 3, wherein(i) the monocyte is a CD11b⁺ monocyte, but is not a dendritic cell whichdifferentiation is controlled by following transcription factors:IFN-regulatory factor 8 (IRF8), nuclear factor interleukin(IL)-3-regulated protein (NFIL3), basic leucine zipper transcriptionalfactor ATF-like 3 (BATF3) or Transcription Factor RelB (NF-KBSubunit)-RELB, Spi 1 Proto-Oncogene (PU/1), recombining binding proteinsuppressor of hairless (RBPJ), IFN-regulatory factor 4 (IRF4) ortranscription factor E2-2 (also known as (TCF4); (ii) the differentiatedmonocyte is selected from the group consisting of a macrophage, anactivated macrophage, preferably a CD11b⁺ macrophage, more preferably aCD11b⁺ CD16⁺ macrophage, CD11b⁺ CD32⁺ macrophage, CD11b⁺ CD64⁺macrophage, CD11b⁺ CD68⁺ macrophage, preferably a CD11b⁺ CD86⁺ M1macrophage, preferably producing iNOS and/or secreting interleukin 12(IL-12) or preferably CD11b⁺ CCR2⁺ M2 macrophage, CD11b⁺ CD204⁺ M2macrophage, CD11b⁺ CD206⁺ M2 macrophage, CD11b⁺ CD204⁺ CD206⁺ M2macrophage, CD11b⁺ Mayor Histocompatibility Complex II⁺ (MHCII⁺) (low orhi expression) M2 macrophage, CD11b⁺ CD200R⁺ M2 macrophage, CD11b⁺CD163⁺ M2 macrophage or activated macrophage producing arginase and/orsecreting interleukin 10 (IL-10); preferably the differentiatedmonocyte-macrophage is not a Lox1⁺, CXCR7⁺ and NRF2⁺ foam cell; (iii)monocyte-macrophage or activated monocyte-macrophage expressing of atleast one chemokine receptor, or at least one growth factor receptor;(iv) the lymphocyte is selected from the group consisting of a CD3⁺ andCD4⁺ or CD8⁺ T lymphocyte, or a CD19⁺, CD20⁺, CD21⁺, CD19⁺ CD20⁺, CD19⁺CD21⁺, CD20⁺ CD21⁺, or CD19⁺ CD20⁺ CD21⁺ B lymphocyte, and a naturalkiller (NK) cell; or (v) the granulocyte is selected from the groupconsisting of a neutrophil, an eosinophil and a basophil.
 5. Theisolated targeted delivery system of claim 4, wherein the activatedmacrophage: (i) is producible by in vitro incubation of a monocyte ormacrophage with a factor capable of altering expression markers onmacrophages; (ii) is characterized by expression of at least one offollowing antigens: CD64, CD86, CD16, CD32, high expression of WICKand/or production of iNOS and/or IL-12; (iii) is producible by in vitroincubation of a monocyte or macrophage with a factor capable of inducingthe ability of the macrophage to phagocytose; (iv) is characterized byexpression of at least one of following antigens: CD204, CD206, CD200R;CCR2, transferrin receptor (TfR), CXC-motive chemokine receptor 4(CXCR4), CD163, and/or T cell immunoglobulin-domain and mucin-domain 2(TIM-2), and/or show low expression of MHCII; (v) has the ability tophagocytose; and/or (vi) is capable of cytokine secretion, or productionof inducible nitric oxide synthetase (iNOS) or other pro-inflammatorycompounds, arginase or other immunosuppressive/anti-inflammatorycompounds.
 6. The targeted delivery system according to claim 5,wherein: (i) the M1 inducer is selected from the group consisting ofLPS, INF-γ, and viral and bacterial infection; or (ii) the M2 inducer isselected from the group consisting of IL-4, IL-10, IL-13, immune complexof an antigen and antibody, IgG, heat activated gamma-globulin,glucocorticosteroid, TGF-β, IL-1R, CCL-2, IL-6, M-CSF, PPARγ agonist,Leukocyte inhibitory factor, adenosine, helminth and fungal infection.7. The isolated targeted delivery system of claim 4, wherein themonocyte-macrophage cell: (i) is producible from a CD34⁺ hematopoieticprecursor cell; (ii) is producible by in vitro incubation ofmonocytes/monocyte-macrophage with at least one inducer; (iii) ischaracterized by expression of at least one of the following antigens:TfR⁺, CD163⁺, TIM-2⁺, CD14⁺, CD16⁺, CD33⁺, and/or CD115⁺; (iv) ischaracterized by expression of at least one of the following antigens:TfR⁺, CD163⁺, TIM-2⁺, CXCR4⁺, CD14⁺, and/or CD16⁺; and/or (v) has theability to phagocytose.
 8. The targeted delivery system according toclaim 7, wherein: (i) the M1 inducer is selected from the groupconsisting of LPS, INF-γ, or viral or bacterial infection; (ii) the M2inducer is selected from the group consisting of IL-4, IL-10, IL-13,immune complex of an antigen and antibody, IgG, heat activatedgamma-globulins, Glucocorticosteroids, TGF-β, IL-1R, CCL-2, IL-6, M-CSF,PPARγ agonist, Leukocyte inhibitory factor, cancer-conditioned medium,cancer cells, adenosine and helminth or fungal infection.
 9. Theisolated targeted delivery system of claim 4, wherein the lymphocyte:(i) is obtainable from blood, spleen, or bone marrow or is produciblefrom a CD34⁺ precursor cell; (ii) is an immunologically competentlymphocyte; (iii) expresses antigen specific T cell receptors; and/or(iv) is characterized by expression of at least one of the followingantigens: (a) CD3⁺ and CD4⁺ or CD8⁺ or (b): CD19⁺, CD20⁺, CD21⁺, CD19⁺CD20⁺, CD19⁺ CD21⁺, CD20⁺ CD21⁺, or CD19⁺ CD20⁺ CD21⁺ antigen.
 10. Theisolated targeted delivery system of claim 4, wherein the granulocyte:(i) is obtainable from blood, spleen or bone marrow or producible from aCD34⁺ precursor cell; (ii) is characterized by expression of at leastone of the following CD66b⁺ and/or CD193⁺; (iii) is a polymorphonuclearleukocyte characterized by the presence of granules in their cytoplasm;and/or (iv) is characterized by expression of at least one of thefollowing: TfR⁺, CD163⁺, TIM-2⁺, and/or CXCR4⁺.
 11. The isolatedtargeted delivery system of claim 4, wherein the NK cell: (i) isobtainable from blood, spleen or bone marrow or producible from a CD34⁺precursor cell; and/or (ii) is characterized by the lack of CD3expression and expression of at least one of the following CD56⁺ and/orCD94⁺, CD158a⁺ CD158f⁺ CD314⁺ CD335⁺.
 12. The isolated targeted deliverysystem of claim 1, wherein the iron binding protein is selected from thegroup consisting of ferritin, haemoglobin, haemoglobin-haptoglobincomplex, hemopexin, transferrin, and lactoferrin.
 13. The isolatedtargeted delivery system according to claim 1, wherein thepharmaceutically active substance is selected from the group consistingof a protein, a nucleic acid, a non-protein non-nucleic acid compoundwith a molecular weight of less than 1.5 kD, an anti-arterioscleroticdrug, an anti-inflammatory drug, a photosensitizing compound, a virus,and a α or ß radiation emitting radioisotope, which also emit a celldamaging amount of γ radiation or a cell damaging amount of α radiation,or a complex of the compound or isotope linked to a nanoparticle. 14.The isolated targeted delivery system according to claim 13, wherein thepharmaceutically active substance is an anticancer drug, wherein theanticancer drug is selected from the group consisting of anapoptosis-inducing drug, an alkylating substance, anti-metabolites,antibiotics, epothilones, nuclear receptor agonists and antagonists, ananti-androgene, an anti-estrogen, a platinum compound, a hormone, aantihormone, an interferon, an inhibitor of cell cycle-dependent proteinkinases (CDKs), an inhibitor of cyclooxygenases and/or lipoxygenases, abiogeneic fatty acid, a biogenic fatty acid derivative, includingprostanoids and leukotrienes, an inhibitor of protein kinases, aninhibitor of protein phosphatases, an inhibitor of lipid kinases, aplatinum coordination complex, an ethyleneimine, a methylmelamine, atriazine, a vinca alkaloid, a pyrimidine analog, a purine analog, analkylsulfonate, a folic acid analog, an anthracendione, a substitutedurea, and a methylhydrazin derivative, an ene-diyne antibiotic, amaytansinoid an auristatine derivate, immune check-point inhibitor, andan inhibitor of tumour-specific protein or marker, preferably aRho-GDP-dissociation inhibitor, more preferably Grp94 or AXL inhibitor.15. The isolated targeted delivery system according to claim 13, whereinthe pharmaceutically active substance is an anticancer drug, wherein theanticancer drug is selected from the group consisting of aacediasulfone, aclarubicine, ambazone, aminoglutethimide,L-asparaginase, azathioprine, banoxantrone, bendamustine, bleomycin,busulfan, calcium folinate, carboplatin, carpecitabine, carmustine,celecoxib, chlorambucil, cis-platin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin dapsone, daunorubicin,dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin,enediynes, epirubicin, epothilone B, epothilone D, estramucin phosphate,estrogen, ethinylestradiole, etoposide, flavopiridol, floxuridine,fludarabine, fluorouracil, fluoxymesterone, flutamide fosfestrol,furazolidone, gemcitabine, gonadotropin releasing hormone analog,hexamethylmelamine, hydroxycarbamide, hydroxymethylnitrofurantoin,hydroxyprogesteronecaproat, hydroxyurea, idarubicin, idoxuridine,ifosfamide, interferon α, irinotecan, leuprolide, lomustine, lurtotecan,mafenide sulfate olamide, mechlorethamine, medroxyprogesterone acetate,megastrolacetate, melphalan, mepacrine, mercaptopurine, methotrexate,metronidazole, mitomycin C, mitopodozide, mitotane, mitoxantrone,mithramycin, nalidixic acid, nifuratel, nifuroxazide, nifuralazine,nifurtimox, nimustine, ninorazole, nitrofurantoin, nitrogen mustards,oleomucin, oxolinic acid, pentamidine, pentostatin, phenazopyridine,phthalyl sulfathiazole, pipobroman, prednimustine, prednisone, preussin,procarbazine, pyrimethamine, raltitrexed, rapamycin, rofecoxib,rosiglitazone, salazosulfapyridine, scriflavinium chloride, semustinestreptozocine, sulfacarbamide, sulfacetamide, sulfachlopyridazine,sulfadiazine, sulfadicramide, sulfadimethoxine, sulfaethidole,sulfafurazole, sulfaguanidine, sulfaguanole, sulfamethizole,sulfamethoxazole, co-trimoxazole, sulfamethoxydiazine,sulfamethoxypyridazine, sulfamoxole, sulfanilamide, sulfaperin,sulfaphenazole, sulfathiazole, sulfisomidine, staurosporin, tamoxifen,taxol, teniposide, tertiposide, testolactone, testosteronpropionate,thioguanine, thiotepa, tinidazole, topotecan, triaziquone, treosulfan,trimethoprim, trofosfamide, UCN-01, vinblastine, vincristine, vindesine,vinblastine, vinorelbine, and zorubicin, preferably selected from thegroup consisting of auristatin, banoxantrone, bendamustine,chlorambucil, chaliceamycin, dynemycin A, maytansine, melphalan,mertansine, and neocazinostatin.
 16. The isolated targeted deliverysystem according to claim 13, wherein the immunomodulatory drugsactivate or inhibit activity of immune cells, preferably theimmunomodulatory drugs are ligands or antagonists of Pattern RecognitionReceptors, particularly Toll-like Receptors, NOD-like receptors (NLR),RIG-I-like receptors (RLR).
 17. The isolated targeted delivery systemaccording to claim 13, wherein the pharmaceutically active substance isan anticancer drug, wherein the anticancer drug is a proliferationinhibiting protein, or an antibody or antibody like binding protein thatspecifically binds to a proliferation promoting protein or a nucleicacid.
 18. The isolated targeted delivery system according to claim 1comprising a pharmaceutically active substance, wherein thepharmaceutically active substance is a hypoxia-activated prodrug. 19.The isolated targeted delivery system according to claim 1 comprising apharmaceutically active substance, wherein the pharmaceutically activesubstance is an antigen or a nucleic acid encoding an antigen.
 20. Theisolated targeted delivery system of claim 1 comprising a label, whereinthe label is selected from the group consisting of a fluorescent dye, afluorescence emitting isotope, a radioisotope, a detectable polypeptideor nucleic acid encoding a detectable polypeptide and a contrast agent.21. The isolated targeted delivery system of claim 1 comprising a label,wherein the label comprises a chelating agent which forms a complex withdivalent or trivalent metal cations.
 22. The isolated targeted deliverysystem of claim 21, wherein the chelating agent is selected from thegroup consisting of 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraaceticacid (DOTA), ethylenediaminetetraacetic acid (EDTA),1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),triethylenetetramine (TETA), iminodiacetic acid,Diethylenetriamine-N,N,N′,N′,N″-pentaacetic acid (DTPA) and6-Hydrazinopyridine-3-carboxylic acid (HYNIC).
 23. The isolated targeteddelivery system of claim 20, wherein the label comprises a contrastagent, wherein the contrast agent comprises a paramagnetic agent. 24.The isolated targeted delivery system of claim 20, wherein the labelcomprises a radioisotope/fluorescence emitting isotope, wherein theradioisotope/fluorescence emitting isotope is selected from the groupconsisting of alpha radiation emitting isotopes, gamma radiationemitting isotopes, Auger electron emitting isotopes, X-ray emittingisotopes, fluorescent isotopes, fluorescence emitting isotopes, as wellas conjugates and combinations of the above with proteins, peptides,small molecular inhibitors, antibodies or other compounds.
 25. Theisolated targeted delivery system of claim 20, wherein the labelcomprises a fluorescent dye, wherein the fluorescence dye is selectedfrom the group consisting of the following classes of fluorescent dyes:Xanthens, Acridines, Oxazines, Cynines, Styryl dyes, Coumarines,Porphines, Metal-Ligand-Complexes, Fluorescent proteins, Nanocrystals,Perylenes and Phtalocyanines as well as conjugates and combinations ofthese classes of dyes.
 26. The isolated targeted delivery systemaccording to claim 20, wherein the label comprises a detectablepolypeptide, wherein the detectable polypeptide is an autofluorescentprotein.
 27. The isolated targeted delivery system according to claim 1,wherein: (i) the bond(s) between the iron binding protein(s) and thepharmaceutically active substance, label or pharmaceutically activesubstance and label comprised in the complex are covalent and/ornon-covalent; and/or (ii) the pharmaceutically active substance, labelor pharmaceutically active substance and label comprised in the complexis entrapped/encapsulated by the iron binding protein or multimersthereof.
 28. Method of preparation of the isolated targeted deliverysystem of claim 1 comprising steps of a) providing purified iron bindingprotein; b) covalently or non-covalently linking a pharmaceuticallyactive substance, label or pharmaceutically active substance and labelto and/or encapsulating a pharmaceutically active substance, label orpharmaceutically active substance and label in an iron binding protein;c) providing a CD45⁺ leukocyte cell; and d1) incubating the CD45⁺leukocyte cell in the presence of the iron binding protein produced instep b) until the CD45⁺ leukocyte cell is at least partially loaded withthe complex of the iron binding protein and the a pharmaceuticallyactive substance, label or pharmaceutically active substance and labelproduced in step b); and/or d2) incubating CD45⁺ leukocyte cell in thepresence of the label until the CD45⁺ leukocyte cell is at leastpartially labelled with the label.
 29. The isolated targeted deliverysystem of claim 1 for use as a medicament or diagnostic.
 30. Apharmaceutical composition comprising the isolated targeted deliverysystem of claim 1 and a pharmaceutically acceptable carrier and/orsuitable excipient(s).
 31. The isolated targeted delivery system ofclaim 1 for use in preventing, treating or diagnosing a tumour, aninflammatory disease or ischemic areas, or for prophylactic oftherapeutic vaccination, in particular to prevent or treat an infectiousdisease or cancer.